The combination of a neprilysin inhibitor (sacubitril) and angiotensin-II receptor blocker (valsartan) attenuates glomerular and tubular injury in the Zucker Obese rat

Fig. 1. Ambulatory blood pressure was monitored periodically utilizing radio-telemetric transmitters. Mean arterial pressure (MAP) was recorded prior to the beginning of treatment and after 3, 5, 7 and 9 weeks of treatment during the a light and b dark cycles. Compared to ZLC, MAP increased throughout the course of treatment in ZOC. After 9 weeks of treatment MAP was significantly reduced in ZOSV during the light and dark periods compared to ZOC (p < 0.05 indicated by the dagger symbol). n = 4, 4, 4, 3 and 4 for ZLC, ZOC, ZOSV, ZOV and ZOH, respectively

Fig. 2. Sacubitril/valsartan improves filtration barrier injury in the Zucker Obese (ZO) rat. a Measures of plasma cystatin c, a surrogate marker of glomerular filtration rate. b Ratio of plasma creatinine to cystatin c, which is inversely associated with kidney injury. n = 6–9 for each treatment group. c Mesangial expansion is reduced by combination and monotherapies. Representative micrographs of PAS-stained cortical glomeruli. All colors except hot pink PAS stain were filtered. The small insets in the lower right corner of each representative micrograph show the original raw unfiltered PAS-stained images. Scale bars equal 50 μm. n = 5 for each treatment group, except for hydralazine (n = 7). Bar graphs show quantitative analysis of PAS stain in the glomerular mesangium. Symbols: *p < 0.05 versus ZLC; †p < 0.05 versus ZOC; Δp < 0.05 versus ZOSV; §p < 0.05 versus ZOH; αindicates p < 0.05 vs ZLC by two tailed T-test; βp < 0.05 versus ZOC by two-tailed T-test

Fig. 3. Sacubitril/valsartan improves ultrastructure of the glomerular filtration barrier. a Representative micrographs show normal ultrastructure of the glomerular podocyte and adjacent slit pores in ZLC kidneys. b ZOC exhibited profound loss of endothelial fenestrae along with podocyte effacement and loss of slit pore diaphragm which were partially restored by sac/val treatment. Podocyte foot processes were markedly effaced and flattened in b ZOC, d ZOV and e ZOH, however they appeared more normal in c ZOSV. All images are ×2000 magnification; scale bar = 1 μm. f–g Suppression of nephrin and podocin gene expression is rescued in ZOSV, but not ZOV or ZOH. Bar graphs show nephrin and podocin mRNA expression normalized to 18S mRNA and expressed as a fold change from baseline in ZLC group (n = 4 per group). *p < 0.05 versus ZLC; †p < 0.05 versus ZOC; Δp < 0.05 versus ZOSV

Fig. 4. Sacubitril/valsartan improves tubular ultrastructure. Representative TEM images showing ultrastructural remodeling in proximal tubule cells (PTC). a Normal basilar S-1 PTC in ZLC showing electron dense mitochondrial and canalicular morphology. Image also shows adjacent glomerulus with normal podocyte foot processes (POD fp). b Depicts the loss of basilar mitochondria elongation with fragmentation, as well as loss of canalicular morphology in ZOC. c Depicts an improvement in S-1 PTC morphology in ZOSV characterized by elongated mitochondria, but without apparent improvement in canaliculi. d Also demonstrates abnormal S-1 PTC morphology in ZOV, however mitochondrial fragmentation is less apparent than in ZOC and some canaliculi appear normal. e Depicts a marked increase in mitochondrial fragmentation and abnormal chromatin condensation in the S-1 PTC nucleus in ZOH. These remodeling changes indicate marked cellular degeneration in the PTC. All images are ×2000 magnification; scale bar = 1 μm; G, glomerulus; Mt, mitochondria; C, canaliculi

Fig. 5. Sacubitril/valsartan reduces a tubulointerstitial and b periarterial fibrosis in ZO rats. Representative PSR stained images show fibrosis in the renal interstitium in a or surrounding an arteriole in b. Accompanying bar graphs show quantitative analysis of average intensity of PSR staining in a and area of fibrosis normalized to arteriole diameter in b. Data are represented by mean ± SE. n = 5–7 rats per group. Symbols: *p < 0.05 versus ZLC; †p < 0.05 versus ZOC; §p < 0.05 versus ZOH. Scale bars = 50 μm. c–e Bar graphs show Col1α1 and fibronectin mRNA expression normalized to 18S mRNA and expressed as a fold change from baseline in ZLC group (n = 4 per group). *p < 0.05 versus ZLC; †p < 0.05 versus ZOC; Δp < 0.05 versus ZOSV; §p < 0.05 vs ZOH

Fig. 6. Sacubitril/valsartan reduces a glomerular and b tubulointerstitial nitroso-oxidative stress in ZO rats. Representative images of 3-nitrotyrosine immunostaining as a marker for nitroso-oxidative stress with accompanying bar graphs showing quantitation of measures of intensity. Data are represented by mean ± SE. n = 5–7 rats per group. c–e Bar graphs show NOX2, NOX4 and AT1R mRNA expression normalized to 18S mRNA and expressed as a fold change from baseline in ZLC group (n = 4 per group). Symbols: *p < 0.05 versus ZLC; †p < 0.05 versus ZOC

Fig. 7. Treatment effects on the renal resistivity index. Data are represented by mean ± SE in the accompanying bar graph. n = 6–7 rats per group. Symbols: *p < 0.05 versus ZLC; †p < 0.05 versus ZOC; §p < 0.05 versus ZOH