Losartan reduces ensuing chronic kidney disease and mortality after acute kidney injury

Abstract

Acute kidney injury (AKI) is an important risk factor for incident chronic kidney disease (CKD). Clinical studies disclose that ensuing CKD progresses after functional recovery from AKI, but the underlying mechanisms remain illusive. Using a murine model representing AKI-CKD continuum, we show angiotensin II type 1a (AT1a) receptor signaling as one of the underlying mechanisms. Male adult CD-1 mice presented severe AKI with 20% mortality within 2 weeks after right nephrectomy and left renal ischemia-reperfusion injury. Despite functional recovery, focal tubular atrophy, interstitial cell infiltration and fibrosis, upregulation of genes encoding angiotensinogen and AT1a receptor were shown in kidneys 4 weeks after AKI. Thereafter mice manifested increase of blood pressure, albuminuria and azotemia progressively. Drinking water with or without losartan or hydralazine was administered to mice from 4 weeks after AKI. Increase of mortality, blood pressure, albuminuria, azotemia and kidney fibrosis was noted in mice with vehicle administration during the 5-month experimental period. On the contrary, these parameters in mice with losartan administration were reduced to the levels shown in control group. Hydralazine did not provide similar beneficial effect though blood pressure was controlled. These findings demonstrate that losartan can reduce ensuing CKD and mortality after functional recovery from AKI.

Figure 1. AKI induced by nephrectomy followed by contralateral renal ischemia-reperfusion injury leading to development of ensuing CKD.

(a,b) Blood urea nitrogen (BUN) and creatinine plasma levels, (c) urine albumin-creatinine ratio (ACR), and (d) systolic blood pressure (BP) in adult male mice 3 months after sham operation, nephrectomy (NX) and NX followed by 26- or 28-minute ischemia/reperfusion to contralateral kidney (NX+IRI-26 min, NX+IRI-28 min) respectively. N = 10, 20, 18 and 16 for sham, NX, NX+IRI-26 min, and NX+IRI-28 min respectively. *P < 0.05, ^‡P < 0.001.

Figure 2. A murine AKI model shows abnormal renal pathology and ongoing injury after functional recovery.

(a) BUN and creatinine plasma levels in adult male mice subjected to sham operation, NX and NX+IRI. (b) Systolic blood pressure at day 28. (c) Representative images of periodic acid-Schiff (PAS) and Masson’s trichrome stain of kidneys at day 28. Scale bar, 100 μm. (d) Renal glomerular volume at day 28. (e,f) Quantitative polymerase chain reaction (QPCR) for gene expression in kidneys at day 28. AKI marker genes Lcn-2 and Havcr-1 encoded neutrophil gelatinase-associated lipocalin and kidney injury molecule-1 respectively. Pro-fibrotic genes Acta2, Col1a1 and Col3a1 encoded α-smooth muscle actin, collagen I α1 and Collagen III α1 chains respectively. The expression levels were normalized by glyceraldehyde 3-phosphate dehydrogenase (Gapdh). *P < 0.05, ^†P < 0.01, ^‡P < 0.001. N = 6 for each group of sham and NX in each time point; N = 13, 11 and 11 for NX+IRI group at day 2, 14 and 28 respectively.

Figure 3. Activation of renin-angiotensin system (RAS) in repaired kidney after functional recovery from AKI.

(a) QPCR for RAS gene expression in kidneys at day 28 after AKI. Agtr1a, Agt, Ace, Ren1 and Ren2 encoded angiotensin II type 1a (AT1a) receptor, angiotensinogen, angiotensin converting enzyme, Renin 1 and Renin 2 respectively. *P < 0.05. N = 6 for each group of sham and NX; N = 11 for NX+IRI group. (b) Representative confocal images of angiotensinogen staining in kidneys at day 28. Scale bar, 20 μm.

Figure 4. CKD development after AKI in a larger mouse cohort.

(a,b) Time course systolic BP and urine ACR after NX or NX+IRI. (c) Plasma levels of BUN and creatinine at 5 months after surgery. (d) Representative images of Masson’s trichrome stain in kidneys at 5 months after surgery. (e) Quantification of interstitial fibrosis area in Masson’s trichrome stain of kidneys. (f) Representative images of PAS stain of kidneys showing shrunken glomerular tuft with widened Bowman’s space at 5 months after surgery. Scale bar, 100 μm. ^†P < 0.01, ^‡P < 0.001. N was shown in (a,b).

Figure 5. Losartan reduces mortality and ensuing CKD after severe AKI.

(a) Plasma BUN and creatinine were analyzed 14 and 27 days after IRI or sham surgery and then the mice were administered water vehicle, losartan, or hydralazine since 1 month after NX+IRI. NX only mice served as the sham control. All mice were sacrificed for analyses 5 months after surgery. N for each group at each time point was shown in (e). (b) Plasma levels of BUN and creatinine 14 days after surgery. (c) Plasma levels of BUN and creatinine 27 days after surgery. N = 32 in NX group, and 39 for each group of NX+IRI in (b,c). Each symbol represents the data from each mouse. (d) Time course systolic BP. NX versus other 3 groups at 1 month; NX+IRI versus other 3 groups at 2, 3, 4, and 5 month. (e) The percentage of survival. (f) Time course urine ACR. (g) Plasma levels of BUN and creatinine at 5 month when N = 32 (NX), 27 (NX+IRI), 37 (Losartan) and 33 (Hydralazine) as shown in (e). *P < 0.05, ^†P < 0.01, ^‡P < 0.001.

Figure 6. Losartan attenuates fibrosis in ensuing CKD after AKI.

(a,b) Representative images and quantification of interstitial fibrosis area in kidneys by Masson’s trichrome stain at 5 months after surgery and specific treatment when N = 32 (NX), 27 (NX+IRI), 37 (Losartan) and 33 (Hydralazine) as shown in Fig. 5e. (c,d) Renal glomerular volume, glomerulosclerosis and the percentage of glomeruli with shrunken tufts and widened Bowman’s space. *P < 0.05, ^‡P < 0.001.

Figure 7. Losartan attenuates pro-fibrotic genes in kidney after AKI.

QPCR of renal pro-fibrotic genes (a) transforming growth factor-b1 (Tgfb1), (b) Acta2, (c) Col1a1, and (d) Col3a1 at 5 months. N = 32 (NX), 27 (NX+IRI), 37 (Losartan) and 33 (Hydralazine) as shown in Fig. 5e. *P < 0.05, ^†P < 0.01, ^‡P < 0.001.