A rat model of SHPT with bone abnormalities in CKD induced by adenine and a high phosphorus diet.

The study of parathyroid hyperplasia with bone disease as a critical manifestation of chronic kidney disease-mineral and bone disorders (CKD-MBDs) is challenging due to the lack of a suitable research model. Here, we established a rat model with secondary hyperparathyroidism (SHPT) and bone disease induced by adenine and a high phosphorous diet and analyzed the skeletal characteristics. We performed blood analysis, emission computed tomography (ECT), dual energy X-ray absorptiometry (DEXA), micro-computed tomography (micro-CT), bone histomorphometry, and bone mechanical tests. The CKD rats with SHPT induced by adenine and a high phosphorus diet showed severe abnormalities in calcium and phosphorus metabolism and exhibited parathyroid hyperplasia. The bone mineral density (BMD) of femurs and lumbar vertebrae was significantly lower in the CKD rats than in the control (CTL) rats. The cortical and trabecular bone parameters of femurs showed significant bone loss. In addition, we found decreases in ultimate force, work to failure, stiffness, and elastic modulus in the CKD rats. In conclusion, our findings demonstrated that the CKD rats with SHPT induced by adenine and a high phosphorus diet may serve as a useful model for skeletal analysis in CKD with SHPT.

[Effect of Cordyceps sinensis powder on renal oxidative stress and mitochondria functions in 5/6 nephrectomized rats].

OBJECTIVE: To observe the effect of Cordyceps sinensis (CS) powder on renal oxidative stress and mitochondria functions in 5/6 nephrectomized rats, and to primarily explore its possible mechanisms. METHODS: Totally 30 male Sprague-Dawley rats were divided into the sham-operation group, the model group, and the treatment group by random digit table, 10 in each group. A chronic kidney disease (CKD) rat model was prepared by one step 5/6 nephrectomy. Rats in the treatment group were intragastrically administered with CS powder solution at the daily dose of 2 g/kg, once per day. Equal volume of double distilled water was intragastrically administered to rats in the sham-operation group and the model group. All medication lasted for 12 weeks. The general condition of rats, their body weight, blood pressure, 24 h proteinuria, urinary N-acetyl-ß-D-glucosaminidase (NAG), serum creatinine (SCr) , and blood urea nitrogen (BUN) were assessed before surgery, at week 2, 4, 6, 8, 10, and 10 after surgery. Pathological changes of renal tissues were observed under light microscope. Morphological changes of mitochondria in renal tubular epithelial cells were observed under transmission electron microscope. Activities of antioxidant enzymes including reduced glutathione (GSH), manganese superoxide dismutase (MnSOD), and malondialdehyde (MDA) in fresh renal tissue homogenate were detected. Mitochondria of renal tissues were extracted to detect levels of mitochondrial membrane potential and changes of reactive oxygen species (ROS). And expressions of cytochrome-C (Cyto-C) and prohibitin in both mitochondria and cytoplasm of the renal cortex were also measured by Western blot. RESULTS: (1) Compared with the sham-operation group, body weight was significantly decreased at week 2 (P <0. 01), but blood pressure increased at week 4 (P <0. 05) in the model group. Compared with the model group, body weight was significantly increased at week 12 (P <0. 01), but blood pressure decreased at week 8 (P < 0. 01) in the treatment group. (2) Compared with the sham-operation group, 24 h proteinuria, urinary NAG, blood SCr and BUN significantly increased in the model group (all P <0. 01). Compared with the model group, blood and urinary biochemical indices all significantly decreased in the treatment group (all P <0. 01). (3) Results of pathological renal scoring: Glomerular sclerosis index, scoring for tubulointerstitial fibrosis, degree of tubulointerstitial inflammatory infiltration were all obviously higher in the model group than in the sham-operation group (all P <0. 01). All the aforesaid indices were more obviously improved in the treatment group than in the model group (all P <0. 01). (4) Compared with the sham-operation group, activities of MnSOD and GSH-Px were significantly reduced, but MDA contents obviously increased in the renal cortex of the model group (all P <0. 01). Compared with the model group, activities of MnSOD and GSH-Px obviously increased (P <0. 05, P <0. 01), but MDA contents obviously decreased in the renal cortex of the treatment group (P <0. 01). (5) Compared with the sham-operation group, the mitochondrial membrane potential significantly decreased, but ROS levels significantly increased in the model group (all P <0.01). Compared with the model group, mitochondrial transmembrane potential increased in the treatment group, thereby inhibiting the tendency of increased production of ROS (both P < 0. 01). (6) Results of Western blot showed that, compared with the sham-operation group, expression levels of mitochondrial Cyto-C and Prohibitin were significantly reduced in the renal cortex (P <0. 01), but significantly elevated in the cytoplasm of the model group (P <0. 01). Compared with the model group, each index was obviously improved in the treatment group with statistical difference (P <0. 05, P <0. 01). CONCLUSION: CS powder had renal protection, and its mechanism might partially depend on in- hibition of oxidative stress and protection for mitochondria.

Inhibition of TGF-ß1/Smad signal pathway is involved in the effect of Cordyceps sinensis against renal fibrosis in 5/6 nephrectomy rats.

UNLABELLED: The present study aimed to investigate the effects of Cordyceps sinensis on renal fibrosis and its possible mechanisms. Sprague-Dawley rats were randomly divided into three groups: sham operation (SHAM) group, 5/6 subtotal nephrectomy (SNx) untreated group, and 5/6 subtotal nephrectomy treated with C. sinensis (2.0 g/kg d) (CS) group. Rats were studied 12 weeks after the surgery, and the CS group presented with significantly lower proteinuria, and better renal function compared with the SNx group (p<0.05). Pathological study showed that the glomerulosclerosis tubulointerstitial injury score was significantly reduced in the CS group compared with the SNx group. Furthermore, the mRNA expression of TGF-ß1, Smad2 and Smad3 and the protein expression of TGF-ß1, TßRI, TßRII and p-Smad2/3 were attenuated by the C. sinensis treatment. In constrast, the mRNA and protein expression of Smad7 was upregulated. Furthermore, the expression of α-SMA and FSP1 was also significantly attenuated, accompanied by the increasing expression of E-cadherin, suggesting the inhibition of the epithelial-mesenchymal transition (EMT). IN CONCLUSION: C. sinensis exerted its antifibrotic effect on the SNx rats through the inhibition of the TGF-ß1/Smad pathway.

Pirfenidone inhibits macrophage infiltration in 5/6 nephrectomized rats.

Tubulointerstitial macrophage infiltration is a hallmark of chronic kidney disease involved in the progression of renal fibrosis. Pirfenidone is a newly identified antifibrotic drug, the potential mechanism of which remains unclear. The aim of this study was to investigate the effects of pirfenidone on M1/M2 macrophage infiltration in nephrectomized rats. Nephrectomized rats were treated with pirfenidone by gavage for 12 wk. Twenty-four hour urinary protein, N-acetyl-ß-D-glycosaminidase (NAG) activity, systolic blood pressure, and C-reactive protein were determined. Paraffin-embedded sections were stained for CD68, CCR7, and CD163 macrophages. Monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1α (MIP-1α), as well as M1 and M2 macrophages secretory markers, were evaluated by real-time RT-PCR and Western blotting analysis. Pirfenidone significantly improved the elevated proteinuria and NAG activity from week 2 onward after surgery. Pirfenidone attenuated interstitial fibrosis and decreased expression of fibrotic markers including transforming growth factor-ß(1), connective tissue growth factor, α-smooth muscle actin, fibronectin, and fibroblast-specific protein-1. Pirfenidone significantly decreased the infiltrating macrophages. The number of M1 and M2 macrophages was significantly lower after pirfenidone treatment. MCP-1 and MIP-1α were increased in nephrectomized rats at mRNA and protein levels. Pirfenidone treatment significantly inhibited their expression. The TNF-α, IL-6, and nitric oxide synthases-2 expressed by M1 macrophages were increased in nephrectomized rats, and pirfenidone significantly attenuated their expression. Pirfenidone treatment also significantly decreased arginase-1, dectin-1, CD206, and CD86 expressed by M2 macrophages. Thus pirfenidone inhibits M1 and M2 macrophage infiltration in 5/6 nephrectomized rats, which suggests its efficacy in the early and late periods of renal fibrosis.