Adaptation mechanism of three Impatiens species to different habitats based on stem morphology, lignin and MYB4 gene.

BACKGROUND: Impatiens is an important genus with rich species of garden plants, and its distribution is extremely extensive, which is reflected in its diverse ecological environment. However, the specific mechanisms of Impatiens' adaptation to various environments and the mechanism related to lignin remain unclear. RESULTS: Three representative Impatiens species,Impatiens chlorosepala (wet, low degree of lignification), Impatiens uliginosa (aquatic, moderate degree of lignification) and Impatiens rubrostriata (terrestrial, high degree of lignification), were selected and analyzed for their anatomical structures, lignin content and composition, and lignin-related gene expression. There are significant differences in anatomical parameters among the stems of three Impatiens species, and the anatomical structure is consistent with the determination results of lignin content. Furthermore, the thickness of the xylem and cell walls, as well as the ratio of cell wall thickness to stem diameter have a strong correlation with lignin content. The anatomical structure and degree of lignification in Impatiens can be attributed to the plant's growth environment, morphology, and growth rate. Our analysis of lignin-related genes revealed a negative correlation between the MYB4 gene and lignin content. The MYB4 gene may control the lignin synthesis in Impatiens by controlling the structural genes involved in the lignin synthesis pathway, such as HCT, C3H, and COMT. Nonetheless, the regulation pathway differs between species of Impatiens. CONCLUSIONS: This study demonstrated consistency between the stem anatomy of Impatiens and the results obtained from lignin content and composition analyses. It is speculated that MYB4 negatively regulates the lignin synthesis in the stems of three Impatiens species by regulating the expression of structural genes, and its regulation mechanism appears to vary across different Impatiens species. This study analyses the variations among different Impatiens plants in diverse habitats, and can guide further molecular investigations of lignin biosynthesis in Impatiens.

Effects of cell morphology, physiology, biochemistry and CHS genes on four flower colors of Impatiens uliginosa.

Introduction: Flower color is one of the important ornamental traits in the plants, which plays an active role in attracting pollinators to pollinate plants and reproduce their offspring. The flower color of Impatiens uliginosa is rich, there are four main flower colors in nature: deep red, red, pink, and white. However, it remains unclear whether on four different flower colors mechanism of I. uliginosa. Methods: We investigate colorimetric measurement, observation of epidermal cells, cellular pH determination, extraction and determination of total anthocyanins and flavonoid, semi-quantitative determination of pigment components, and gene cloning and qRT-PCR of CHS genes to study four flower colors of I. uliginosa. Results: The L* and b* values were the highest in white flower, while the a* values were the highest in pink flower. The same shape of epidermal cells was observed in different flower colors, which was all irregular flat polygons, and there were partial lignification. Their cellular pH values were weakly acidic, while the pH values of the deep red flower was the highest and the white flower was the lowest. The highest pigment content of the four flower colors was total anthocyanin content. And malvidin-3-galactosidechloride (C23H25ClO12), cyanidin-3-O-glucoside (C21H21O11) and delphinidin (C15H11O7) were the main pigment components affecting the color of four different flower colors. The anthocyanin synthesis gene IuCHS was expressed in four flowers, and all three copies of it had the highest expression level in pink flower and the lowest expression level in white flower. Discussion: These results revealed the influence of main internal factors on four different flower colors of I. uliginosa, and provided a basis for further understanding of the intracellular and molecular regulatory mechanisms of flower color variation, and laid a foundation for the improvement of flower color breeding of Impatiens.

The cellular and molecular basis of the spur development in Impatiens uliginosa.

The nectar spur is an important feature of pollination and ecological adaptation in flowering plants, and it is a key innovation to promote species diversity in certain plant lineages. The development mechanism of spurs varies among different plant taxa. As one of the largest angiosperm genera, we have little understanding of the mechanism of spur development in Impatiens. Here, we investigated the initiation and growth process of spurs of Impatiens uliginosa based on histology and hormone levels, and the roles of AUXIN BINDING PROTEIN (ABP) and extensin (EXT) in spur development were explored. Our results indicate that the spur development of I. uliginosa is composed of cell division and anisotropic cell elongation. Imbalances in spur proximal-distal cell division lead to the formation of curved structures. Endogenous hormones, such as auxin and cytokinins, were enriched at different developmental stages of spurs. IuABP knockdown led to an increase in spur curves and distortion of morphology. IuEXT knockdown resulted in reduced spur length and loss of curve and inner epidermal papillae structures. This study provides new insights into the mechanism of spur development in core eudicots.

De novo transcriptome sequencing of Impatiens uliginosa and the analysis of candidate genes related to spur development.

BACKGROUND: Spur, a structure capable of producing and storing nectar, not only plays a vital role in the pollination process but also promotes the rapid diversification of some plant lineages, which is considered a key innovation in plants. Spur is the focus of many studies, such as evolution and ecological hypothesis, but the current understanding of spur development is limited. High-throughput sequencing of Impatiens uliginosa was carried out to study the molecular mechanism of its spur development, which is believed to provide some insights into the spur development of Impatiens. RESULTS: Transcriptomic sequencing and analysis were performed on spurs and limbs of I. uliginosa at three developmental stages. A total of 47.83 Gb of clean data were obtained, and 49,716 unigene genes were assembled. After comparison with NR, Swiss-Prot, Pfam, COG, GO and KEGG databases, a total of 27,686 genes were annotated successfully. Through comparative analysis, 19,356 differentially expressed genes were found and enriched into 208 GO terms and 146 KEGG pathways, among which plant hormone signal transduction was the most significantly enriched pathway. One thousand thirty-two transcription factors were identified, which belonged to 33 TF families such as MYB, bHLH and TCP. Twenty candidate genes that may be involved in spur development were screened and verified by qPCR, such as SBP, IAA and ABP. CONCLUSIONS: Transcriptome data of different developmental stages of spurs were obtained, and a series of candidate genes related to spur development were identified. The importance of genes related to cell cycle, cell division, cell elongation and hormones in spur development was clarified. This study provided valuable information and resources for understanding the molecular mechanism of spur development in Impatiens.

Aureimonas psammosilene sp. nov., isolated from the roots of Psammosilene tunicoides.

One motile strain designated, YIM DR1026T was isolated from the roots of Psammosilene tunicoides collected from Gejiu, Yunnan province, China. The cells of strain YIM DR1026T were Gram-negative and short-rod shaped. Phylogenetic analyses based on 16S rRNA gene sequences indicated that strain YIM DR1026T was a member of the genus Aureimonas and closely related to Aureimonas rubiginis (96.7%). DNA-DNA relatedness values between strain YIM 1026T and Aureimonas rubiginis BCRC 80440T was 38.2 ± 1.5%. The ANI value between YIM DR1026T and other Aureimonas members were below the cut-off level (95-96%) recommended as the average nucleotide identity (ANI) criterion for interspecies identity. Strain YIM DR1026T grew at 4-30 °C (optimum 28 °C), pH 4.0-9.0 (optimum pH 6.0-7.0) and tolerated NaCl (w/v) up to 1% (optimum 0%). Q-10 was sole the respiratory ubiquinone present in YIM DR1026T. Polar lipids of strain YIM DR1026T were phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylcholine, sulfoquinovosyldiacylglycerol, unidentified aminolipid and unidentified polar lipid. The genomic G + C content was 64.6 mol%. The major fatty acids were C18:1ω7c, C16:0 and summed feature 3 (C16:1ω7c/C16:1ω6c). Based on phenotypic, phylogenetic, chemotaxonomic and genome comparison, strain YIM DR1026T represents a novel species of the genus Aureimonas, for which the name Aureimonas psammosilene sp. nov. is proposed. The type strain is YIM DR1026T (= KCTC 42691T = NBRC 112412T).

Nocardia zhihengii sp. nov., an actinobacterium isolated from rhizosphere soil of Psammosilene tunicoides.

A Nocardia-like actinobacterial strain, designated YIM TG2190T, was isolated from rhizosphere soil of Psammosilene tunicoides collected from Gejiu, Yunnan province, China. The cells of strain YIM TG2190T were observed to be Gram-stain positive and non-motile. The strain forms extensively branched substrate mycelia that fragments into rod-shaped elements. The 16S rRNA gene sequence analysis showed that strain YIM TG2190T is closely related to Nocardia nova (97.5%), Nocardia jiangxiensis (97.1%) and Nocardia miyunensis (96.8%). Growth occurs at 4-30 °C (optimum 28 °C), pH 6.0-8.0 (optimum pH 7.0) and the strain can tolerate NaCl (w/v) up to 3% (optimum 0-1%). The cell walls were found to contain meso-diaminopimelic acid. The whole-cell sugars were identified as glucose, mannose, ribose, galactose, arabinose and fucose. The polar lipids were identified as diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol mannosides, phosphatidylglycerol and an unidentified phospholipid. The menaquinones detected were MK-9 (H2) and MK-8 (H4). The major fatty acids (> 5%) were found to be C16:0 (33.9%), summed feature 3 (21.7%), C18:0 10-methyl TBSA (13.7%) and C18:1ω9c (7.0%). The DNA G+C content was determined to be 61.1 mol%. DNA-DNA relatedness between the strain YIM TG2190T and N. nova CGMCC 4.1705T, N. jiangxiensis CGMCC 4.1905T and N. miyunensis CGMCC 4.1904T were 46.9 ± 2.6, 36.8 ± 1.3, and 35.7 ± 2.6%, respectively, values which are less than the threshold value (70%) for the delineation of prokaryotic genomic species. The phenotypic, chemotaxonomic and phylogenetic data indicates that strain YIM TG2190T represents a novel species of the genus Nocardia, for which the name Nocardia zhihengii sp. nov. is proposed. The type strain is YIM TG2190T (=KCTC 39596T = DSM 100515T).

Allostreptomyces psammosilenae gen. nov., sp. nov., an endophytic actinobacterium isolated from the roots of Psammosilene tunicoides and emended description of the family Streptomycetaceae [Waksman and Henrici (1943)AL] emend. Rainey et al. 1997, emend. Kim et al. 2003, emend. Zhi et al. 2009.

A Gram-stain-positive actinobacterium, designated strain YIM DR4008T, was isolated from the root sample of Psammosilene tunicoides collected from Lijiang, Yunnan, China. Strain YIM DR4008T could grow at temperatures ranging from 10 to 50 °C (optimum 28-30 °C), at pH 5.0-11.0 (optimum pH 7.0) and in the presence of up to 4 % (w/v) NaCl. Sequence analysis of the 16S ribosomal RNA gene revealed that strain YIM DR4008T shared highest similarity (95.0 %) with Streptomyces griseoplanus NBRC 12779T and <95 % similarity with other known members of the genera Streptomyces, Kitasatospora and Streptacidiphilus. The diagnostic cell-wall diamino acid of strain YIM DR4008T was found to be ll-diaminopimelic acid. The whole-cell hydrolysates contained a major amount of galactose and mannose along with a small proportion of fucose, glucose, rhamnose and ribose. The polar lipids consisted of diphosphatidylglycerol, phosphatidylinositol mannosides and three unidentified phospholipids. The respiratory menaquinones were MK-9(H6) and MK-9(H8), while the major cellular fatty acids (>10 %) were anteiso-C15 : 0, C16 : 0, iso-C16 : 0, iso-C15 : 0 and anteiso-C17 : 0. The genomic DNA G+C content was determined to be 75.3 mol%. Based on the phenotypic, chemotaxonomic and molecular characteristics, strain YIM DR4008T is proposed to be recognized as a novel species of a new genus in the family Streptomycetaceae, with the name Allostreptomyces psammosilenae gen. nov., sp. nov. The type strain of the type species is YIM DR4008T (=DSM 42178T=CGMCC 4.7247T). An emended description of the family Streptomycetaceae is also provided.

Nocardioides intraradicalis sp. nov., isolated from the roots of Psammosilene tunicoides W. C. Wu et C. Y. Wu.

A Gram-stain-positive, non-spore-forming and non-motile strain, designated YIM DR1091T, was isolated from the roots of Psammosilene tunicoides W. C. Wu et C. Y. Wu collected from Gejiu, Yunnan, China. The taxonomic position of strain YIM DR1091T was investigated by a polyphasic approach. Phylogenetic analyses based on 16S rRNA gene sequences indicated that strain YIM DR1091T is a member of the genus Nocardioides. Strain YIM DR1091T was closely related to Nocardioides pyridinolyticus OS4T, Nocardioides caricicola YC6903T, Nocardioides hankookensis DS-30T and Nocardioides aquiterrae GW-9T, with which it shared pairwise 16S rRNA gene sequence similarities of 97.6, 97.5, 97.2 and 97.2 %, respectively. Mean DNA-DNA relatedness values between strain YIM DR1091T and related type strains N. pyridinolyticus JCM 10369T, N. caricicola JCM 17686T, N. hankookensis JCM 15302T and N. aquiterrae JCM 11813T were 44.9±1.7, 50.2±1.3, 46.8±0.9 and 43.0±0.2 %, respectively. The respiratory menaquinone for strain YIM DR1091T was MK-8(H4) while the major fatty acids (>5 %) were iso-C16 : 0, C17 : 1ω8c, C17 : 0, iso-C15 : 0 and iso-C14 : 0. The polar lipids were diphosphatidylglycerol, phosphatidylglycerol and three unidentified phospholipids. Whole-cell hydrolysates contained mannose, ribose, glucose and galactose, along with ll-diaminopimelic acid as the diagnostic diamino acid in the peptidoglycan. The DNA G+C content was 74.6 mol%. Phenotypic, phylogenetic and chemotaxonomic data indicated that strain YIM DR1091T represents a novel species of the genus Nocardioides, for which the name Nocardioides intraradicalis sp. nov. is proposed. The type strain is YIM DR1091T (=JCM 30632T=CGMCC4.7251T).

Streptomyces zhihengii sp. nov., isolated from rhizospheric soil of Psammosilene tunicoides.

An actinomycete strain, designated YIM T102(T), was isolated from the rhizospheric soil of Psammosilene tunicoides W. C. Wu et C. Y. Wu collected from Lijiang, Yunnan Province, China. The taxonomic position of the new isolate was investigated by a polyphasic approach. Phylogenetic analyses based on 16S rRNA gene sequences indicated that strain YIM T102(T) belongs to the genus Streptomyces. Strain YIM T102(T) was most closely related to Streptomyces eurocidicus NRRL B-1676(T) with a pairwise 16S rRNA gene sequence similarity of 98.9 %. However, DNA-DNA relatedness value between strain YIM T102(T) and S. eurocidicus NBRC 13491(T) was found to be 37.8 ± 1.8 %. The menaquinone composition detected for strain YIM T102(T) was MK-9 (H6) and MK-9 (H8), while the major fatty acids were summed feature 4 (38.0 %), anteiso-C15:0 (13.1 %), iso-C16:0 (10.1 %), summed feature 3 (9.8 %) and C16:0 (9.0 %) and iso-C15:0 (5.2 %). The whole-cell hydrolysates contained galactose, glucose, ribose and mannose, along with LL-diaminopimelic acid as the diagnostic diamino acid in the peptidoglycan. The DNA G+C content was 70.7 mol%. Strain YIM T102(T) also exhibited antagonistic activity against Alternaria alternata, Alternaria brassicae and Colletotrichum nicotianae Averna, based on the findings from the comparative analyses of phenotypic and genotypic characteristics; it is proposed that strain YIM T102 represents a novel species of the genus Streptomyces, for which the name Streptomyces zhihengii sp. nov. is proposed. The type strain is YIM T102(T) (=KCTC 39115(T) = DSM 42176(T) = CGMCC 4.7248(T)).

[The effects of failure of low density lipoprotein receptor expression induced by inflammation on radial artery foam cell formation in patients with end-stage renal disease].

OBJECTIVE: To investigate whether inflammation exacerbates lipid accumulation in the radial arteries of patients with end-stage renal disease (ESRD) and to explore its underlying mechanisms. METHODS: Thirty ESRD patients receiving arteriovenostomy were included. The patients were divided by the plasma level of C-reactive protein into control group (n = 16) and inflamed group (n = 14). Foam cell formation and lipid droplet accumulation were checked by HE staining and Oil red O staining. Tissue inflammation and intracellular cholesterol trafficking correlated proteins were examined by immunohistochemistry or immunofluorescent staining. RESULTS: There were no differences in primary diseases, age, body weight, hemoglobin, total protein, albumin, glucose, lipid profile between the two groups (all P values >0.05). The expressions of tumor necrosis factor α (TNFα) and monocyte chemotactic protein-1 (MCP-1) of the radial artery were increased in the inflamed group. There was significant lipid accumulation in the radial arteries of inflamed group compared to the control group, which was correlated with the increased protein expressions of low density lipoprotein receptor (LDLr), sterol regulatory element binding protein-2 (SREBP-2), and SREBP cleavage-activating protein (SCAP). Confocal microscopy observation showed that inflammation enhanced the translocation of SCAP escorting SREBP-2 from endoplasmic reticulum to Golgi, thereby activating LDLr gene transcription. Further analysis showed that dysregulation of LDLr pathway induced by inflammation was associated with increased protein expression of mTOR (r = 0.733, P < 0.05), especially with the enhanced co-expression of mTOR and SREBP-2(P < 0.05). CONCLUSION: Inflammation accelerates the progression of foam cell formation in ESRD patients via dysregulation of LDLr pathway, which might be partly through the activation of mTOR pathway.

Influence of irbesartan on the urinary excretion of cytokines in patients with chronic kidney disease.

BACKGROUND: The non-hemodynamic effects of angiotensin receptor blocker (ARB) in the delay of progression of chronic kidney disease (CKD) remain unclear. In this study, we investigated the influence of irbesartan on the urinary excretion of cytokines in patients with CKD. METHODS: In this randomized perspective clinical trial, different doses of irbesartan (150 mg/d and 300 mg/d) were given to two groups of patients in a cross-over design. Blood pressure (BP), creatinine clearance (Ccr) and 24-hour proteinuria were examined. Urinary excretion of cytokines was determined by human inflammatory cytokine antibody array. A two-fold change in spot intensity was considered significant. RESULTS: Urinary excretion of cytokines (granulocyte colony stimulating factor (GCSF), intercellular cell adhesion molecule-1 (ICAM-1), interferon γ (IFN-γ), interleukin 1ß (IL-1b), IL-2, IL-6, IL-8, IL-11, IL-15 and macrophage inflammatory protein 1d (MIP-1d)) in group B (irbesartan 300 mg/d) was significantly decreased in comparison to group A (irbesartan 150 mg/d) after 8-week treatment. In group A, 8 weeks of treatment induced a two- to nine-fold reduction in urinary cytokine levels (GCSF, GM-CSF, IFN-γ, IL-1a, IL-11, IL-12p40, MCP-2, MIP-1a), while increasing the dosage to 300 mg/d further decreased the excretion of GCSF, GM-CSF, IL-12p40, MCP-2 and MIP-1a by week 18. There was no significant difference in BP or Ccr between the two groups. However, 24-hour proteinuria was significantly reduced in both groups, and in group A the reduction was dose dependent. CONCLUSION: Irbesartan offers additional renoprotection in a dose-dependent manner by reducing pro-inflammatory cytokines excretion in the urine of CKD patients.

Pyloric resection and delayed gastric liquid emptying in rats.

BACKGROUND: Surgical resection of the distal stomach impairs gastric emptying. Generally, pylorus and the antrum are removed in the distal gastrectomy, however, the pylorus is removed individually under specific circumstances. We focus on the relation between the pyloric resection and the gastric liquid emptying. AIMS: The present investigation aimed to explore the pylorectomy how to influence gastric liquid emptying in rats. METHODS: Pylorectomy and end-to-end gastroduodenal anastomosis were conducted in rats. Electrodes were implanted in the gastrointestinal serosal surface near the stoma. Total stomach, proximal stomach, distal stomach and duodenal liquid emptying, myoelectricities in the gastrointestinal tract near the stoma, and structures were examined with scintigraphy, electrode recording in vivo, and electron microscopy, respectively. RESULTS: Delayed total stomach and distal stomach emptying were found in pylorectomy rats (p<0.001). However, there was no difference in the proximal stomach and the duodenal liquid emptying compared to the controls (p>0.05). The myoelectricity of 3-5 cpm (cycles/min) in antrum and 10-12 cpm in duodenum were found in the controls and no retrograde or antegrade myoelectricities were recorded in the duodenum and antrum. High-frequency myoelectricities (tachygastria) were recorded in the antrum near the stoma (p<0.01), the retrograde and antegrade myoelectricities propagating through the stoma were recorded, and the regenerated interstitial cells of Cajal were found in stoma under electron microscope observation in pylorectomy rat. CONCLUSIONS: The gastroduodenal incoordination and abnormal myoelectricity related to impaired contraction in the antrum caused the delayed liquid gastric emptying in pylorectomy rats.

[The effect of Haynaldia villosa v chromosome on the mitochondrial proteome of wheat-H. villosa chromosome substitution line and translocation line].

Wheat-Haynaldia villosa chromosome substitution line (6A/6V) and translocation lines (6DL/6VS, 6AL/6VS) were obtained through hybridization of H. villosa with powdery mildew susceptible cultivated wheat. Substitution line and translocation lines contain V chromosome or the chromosome short arm (VS) of H. villosa. They are resistant to powdery mildew. In this study, mitochondrial proteome changes were analyzed by using substitution line (6A/6V), translocation line (6DL/6VS) as experimental materials in order to studying the effects of V chromosome on the mitochondrial proteome and related to powdery mildew resistance. The results indicated that 16 new mitochondrial protein spots (spot1, 22kDa/PI8.5; spot2, 31 kDa/PI 7.5; spot3, 28 kDa/PI 7.0; spot4, 31 kDa/PI 6.5; spot5, 40 kDa/PI 7.5; spot6, 40 kDa/PI 7.4; spot7, 80 kDa/PI 8.4; spot8, 50 kDa/PI 7.5; spot9, 60 kDa/PI 7.3; spot10, 65 kDa/PI 6.6; spot11, 65 kDa/PI 6.6; spot12, 73 kDa/PI 7.5; spot13, 73 kDa/PI 7.7; spot14, 46 kDa/PI 7.4; spot15, 46 kDa/PI 7.3; spot16, 38 kDa/PI 6.3) were produced and 7 mitochondrial protein spots (spot1, 40 kDa/PI 7.5; spot2, 43 kDa/PI 7.6; spot3, 48 kDa/PI 7.5; spot4, 42 kDa/PI 8.0; spot5, 43 kDa/PI 7.5; spot6, 32 kDa/PI 4.8; spot7, 40 kDa/PI 5.5) were absent in substitution line, 7 new mitochondrial protein spots (spotl, 43 kDa/PI 6.3; spot2, 60 kDa/PI 6.5; spot3, 60 kDa/PI 6.4; spot4, 65 kDa/PI 7.5; spot5, 55 kDa/PI 8.2; spot6, 31 kDa/PI 8.0; spot7, 43 kDa/PI 8.0) were produced and 6 mitochondrial protein spots (spot1', 66 kDa/PI 8.3; spot2', 58 kDa/PI 8.5; spot3', 36 kDa/PI 7.0; spot4', 48 kDa/PI 7.7; spot5', 48 kDa/PI 6.8; spot6', 43 kDa/PI 6.2) were absent in translocation line. These experimental results suggest that V chromosome or VS of H. villosa can obviously lead mitochondrial proteome changed. These changes may be associated with resistant to powdery mildew of substitution line and translocation line.

[Different proteins in mitochondrial proteome of T-type maize cytoplasmic male-sterile line and its maintainer line].

Using T-type maize cytoplasmic male sterile line (T-CMS) and maintainer line as experimental materials, we separated mitochondrial proteins from leaves at seedling, shooting, booting stages, mesocotyl, root and anther at meiosis of pollen mother cell, single-double nucleus stage of pollen grain by two-dimensional electrophoresis with immobilized pH3-10 gradients. About 150 mitochondrial protein spots in seedling leaves, 150 spots in mesocotyls, 150 spots in roots and 100 spots in meiosis anther were observed respectively in this investigation. 6 difference protein spots were identified by MALDI-TOF-MS analysis and NCBI database searching. r40c1 protein was present in mesocotyl of T-CMS and absent in maintainer line. Mature anther-specific protein, DNA-directed RNA polymerase 23kDa subunit, hexokinase II were present and glutathione S-transferase, putative polyprotein were absent in pollen aborted anther of T-CMS. Developmental changes in mitochondrial proteins were found in leaves but no differences were observed in T-CMS and its maintainer line. Obvious differences of mitochondrial proteins were found at single-double nucleus stage anther in T-CMS and maintainer line. These different proteins were considered to be associated to pollen aborted in T-CMS.

Connective tissue growth factor is associated with the early renal hypertrophy in uninephrectomized diabetic rats.

BACKGROUND: Renal hypertrophy has been regarded as the early feature of diabetic nephropathy (DN), which may eventually lead to proteinuria and renal fibrosis. However, the exact mechanism of renal hypertrophy is still unclear. The aim of this study was to investigate the possible association of connective tissue growth factor (CTGF) with renal hypertrophy in uninephrectomized diabetic rats. METHODS: Seventy-two Sprague-Dawley (SD) rats were randomly divided into two groups: control group (group C, n = 32) and diabetic nephropathy (group DN, n = 40). Each group was re-divided into 4 subgroups according to the experimental period. The rats were sacrificed at 1, 2, 4, and 8 weeks respectively after induction of diabetes. Diabetes was induced by intraperitoneal injection of streptozotocin (STZ) after rats had received uninephrectomy. Blood glucose (BG), body weight (BW), 24-h urinary albumin excretion (24hUalb), kidney weight (KW), KW/BW, glomerular tuft area (AG), glomerular tuft volume (VG), proximal tubular area (AT) at each time point, the width of glomerular basement membrane (GBM) and tubular basement membrane (TBM) at week 8 were measured when the rats were sacrificed. Renal expression of CTGF and p27kip1 were detected by immunohistochemical staining. The relationship between CTGF expression and increasing of VG and AT was analyzed. RESULTS: There was a significant increase of 24hUalb, KW, and KW/BW from week 1 onward in diabetic rats compared to those in group C (P < 0.05, respectively), diabetic rats also had a significant increase of AG, VG, and AT from week 1 onward. It was also shown that diabetic rats had a thickening of GBM [(245.7 +/- 103.0) nm vs (121.8 +/- 19.1) nm, P < 0.01] and TBM [(767.7 +/- 331.1) nm vs (293.0 +/- 110.5) nm, P < 0.01] at week 8. There was a weak expression for CTGF and p27kip1 in normal glomeruli and tubuli, while a significant increasing expression of CTGF and p27kip1 was found in glomeruli and tubuli in diabetic kidney from week 1 onward (P < 0.05, respectively), and the extent of CTGF expression was positively correlated with AG (r = 0.92, P < 0.05), VG (r = 0.86, P < 0.05), AT (r = 0.94, P < 0.01) and positively correlated with the expression of p27kip1 (r = 0.96, P < 0.01). CONCLUSION: The expression of CTGF increases in diabetic rat kidney at the early stage, which might be an important mediator of renal hypertrophy through arresting cell cycling.

Connective tissue growth factor-mediated angiotensin II-induced hypertrophy of proximal tubular cells.

BACKGROUND: Cellular hypertrophy is an early, important pathological feature of renal diseases such as diabetic nephropathy and remnant kidney. Recent studies have demonstrated that angiotensin II (AngII) plays a key role in mediating cell hypertrophy. The aim of our work was to explore the role of connective tissue growth factor (CTGF) in mediating AngII-induced tubular cell hypertrophy in vivoandin vitro. METHODS: In an in vivo study, male Sprague-Dawley rats were randomly divided into three groups: control rats, diabetic rats and diabetic rats treated with irbesartan (IRB). The index of kidney hypertrophy (kidney weight/body weight, KW/BW), glomerular tuft area (AG), glomerular tuft volume (VG) and proximal tubular area (AT) were determined. Renal expression for CTGF was detected by immunohistochemical staining. In an in vitro study, the influence of CTGF antisense oligonucleotide (CTGF AS) on AngII-induced CTGF expression and cell hypertrophy was also investigated. RESULTS: In an in vivo study, diabetic rats showed a significant increase of KW/BW, AG, VG, and AT from week 1 onwards compared to normal control, which could be significantly inhibited by using IRB. Furthermore, there was a significantly increasing expression of CTGF in both glomeruli and tubuli in diabetic rats compared to control, and the extent of CTGF expression closely correlated with the severity of renal hypertrophy. Treatment with IRB could markedly inhibit the renal expression of CTGF. In an in vitro study, AngII stimulated the expression of CTGF mRNA and CTGF protein. AngII significantly increased the total protein content in HK2 cells, which was markedly inhibited by co-treatment with CTGF AS. The average cellular diameter determined by scanning electronic microscope showed that the increase of cell size induced by AngII could be significantly inhibited by CTGF AS. Furthermore, flow cytometer study showed that AngII arrested the cell cycle in the G0-G1 phase, which was significantly reversed by treatment with CTGF AS. CONCLUSION: Our data provide both in vivo and in vitroevidence that CTGF is involved in mediating AngII-induced renal hypertrophy.