Kai Hou

TIMP-1 Transgenic Mice Recover From Diabetes Induced by Multiple Low-Dose Streptozotocin

Type 1 diabetes results from autoimmune destruction of the insulin-producing β-cells of pancreatic islets, of which the capacity for self-replication in the adult is too limited to restore following extensive tissue injury. Tissue inhibitor of metalloproteinase (TIMP)-1 inhibits matrix metalloproteinase activity and regulates proliferation and apoptosis of a variety of cells types, depending on the context. Here, we show that overexpression of human TIMP-1 in pancreatic β-cells of transgenic mice counteracts the cytotoxicity and insulitis induced by multiple low-dose streptozotocin (MLDS). Nontransgenic mice developed severe hyperglycemia, hypoinsulinemia, and insulitis 2 weeks after streptozotocin administration and died within 17 weeks. However, MLDS-treated transgenic mice gradually normalized the metabolic parameters and survived. β-Cell mass increased in parallel as a result of enhancement of β-cell replication. Thus, our results have demonstrated for the first time that overexpression of TIMP-1 in β-cells enhances the replication of pancreatic islets β-cells and counteracts type 1 diabetes, indicating that the TIMP-1 gene may be a potential target to prevent, or even reverse, type 1 diabetes.

Autophagy can repair endoplasmic reticulum stress damage of the passive Heymann nephritis model as revealed by proteomics analysis

Membranous nephropathy is a common cause of nephrotic syndrome in adults. Although many mechanisms have been proposed, whole proteomic research is still lacking. We analyzed the passive Heymann nephritis animal model using label-free quantitative proteome technology. Results showed 160 differential proteins between control and PHN model groups at days 14 and 21. The expression level of endoplasmic reticulum stress (ERS)-associated protein GRP78 and GRP94 protein was up-regulated on day 14 or 21, which was confirmed by Western blotting. The results also showed that the autophagy marker LC3 was up-regulated in the models. Furthermore, we used tunicamycin to induce ERS of podocytes in vitro to investigate the mechanism. Results of Western blotting revealed that the expression of GRP78, GRP94, and LC3 was up-regulated, while that of the cytoskeletal protein tubulin-β was down-regulated, and immunofluorescence displayed disordered distribution of tubulin-β. These suggest that ERS plays an important role in podocyte damage. Autophagy can repair the cytoskeleton damage caused by ERS as a protective mechanism. This provides an important basis for a thorough understanding of the mechanism of podocyte damage and the pathogenesis of membranous nephropathy.

Identification of basolateral membrane targeting signal of human sodium-dependent dicarboxylate transporter 3

Sodium‐dependent dicarboxylate transporters (NaDC) include low‐affinity NaDC1 and high‐affinity NaDC3. Despite high similarities structurally and functionally, both are localized to opposite surfaces of renal tubular cells. The molecular mechanisms and localization signals leading to this polarized distribution remain unknown. In this study, distribution of NaDC3 in human kidney tissue was firstly observed by immunohistochemistry and immunofluorescence. Then, EGFP‐fused wild‐type, NH2‐ and COOH‐terminal deletion and point mutants of NaDC3, and chimera between NaDC3 and NaDC1, were generated and transfected into polarized renal cells lines, LLC‐PK1 and MDCK. Their subcellular localizations were analyzed by laser confocal microscopy. Immunolocalization results revealed that NaDC3 was expressed at basolateral membrane of human renal proximal tubular epithelia. Confocal examinations showed that wild‐type NaDC3 was targeted to the basolateral membrane of MDCK and LLC‐PK1. Deletion mutations indicated that the basolateral targeting signal of NaDC3 located within a short sequence AKKVWSARR of its amino‐terminal cytoplasmic domain. Addition of this sequence could redirect apical NaDC1 to the basolateral membrane of LLC‐PK1. Point mutagenesis revealed that mutation of either of two hydrophobic amino acids V and W in this short sequence largely redirected NaDC3 to both apical and basolateral surfaces of LLC‐PK, indicating that the two hydrophobic amino acids are critical for the basolateral targeting of NaDC3. Our studies provide direct evidence of the localization of NaDC3 at the basolateral membrane of human renal proximal tubule cells and identify a di‐hydrophobic amino acid motif VW as basolateral localization signal in the N‐terminal cytoplasmic domain of NaDC3. J.Cell.Physiol.

Knockdown of Fibronectin Induces Mitochondria-Dependent Apoptosis in Rat Mesangial Cells

Extracellular matrix (ECM) expansion and mesangial cell (MC) proliferation are prominent features of most types of glomerulosclerosis. A delicate balance between the ECM and MC regulates cell survival. Increasing evidence shows that a loss of ECM components can cause mitochondrial dysfunction and induce cell apoptosis. It is proposed that directly blocking the synthesis of ECM components could lighten ECM accumulation and suppress cell overproliferation status. Fibronectin, one of the predominant adhesive glycoproteins of the mesangial ECM, provides the survival signal for cells. Its accumulation can be observed in most types of glomerulosclerosis. In this study, angiotensin II-induced fibronectin was suppressed by an RNA interference technique. It is interesting that MC slowly underwent apoptosis after infection with a retrovirus that continuously suppressed fibronectin synthesis. It was found that MC apoptosis occurred in a mitochondria-dependent manner mainly as a result of cytochrome c release and downstream caspase-3 and -9 activation. Furthermore, it was demonstrated that fibronectin knockdown affected mitochondrial handling of Ca(2+) release from the endoplasmic reticulum. Importantly, blocking the inositol 1,4,5-triphosphate receptor with, 3,4,5-trimethoxybenzoate or decreasing Ca(2+) in the ECM with EGTA partially saved the cells from apoptosis. These studies, which explored a new method for simultaneously inhibiting MC proliferation and ECM accumulation, may represent a novel therapeutic approach to glomerulosclerosis.

Membrane topology structure of human high-affinity, sodium-dependent dicarboxylate transporter

High‐affinity, sodium‐dependent dicarboxylate transporter (NaDC3) is responsible for transport of Krebs cycle intermediates and may involve in regulation of aging and life span. Hydropathy analysis predicts that NaDC3 contains 11 or 12 hydrophobic transmembrane (TM) domains. However, the actual membrane topological structure of NaDC3 remains unknown. In this study, confocal immunofluorescence microscopy and membrane biotinylation of epitope‐tagged N and C termini of NaDC3 provide evidence of an extracellular C terminus and an intracellular N terminus, indicating an odd number of transmembrane regions. The position of hydrophilic loops within NaDC3 was identified with antibodies against the loops domains combined with cysteine accessibility methods. A confocal image of membrane localization and transport activity assay of the cysteine insertion mutants show behavior similar to that of wild‐type NaDC3 in transfected HEK293 cells, suggesting that these mutants retain a native protein configuration. We find that NaDC3 contains 11 transmembrane helices. The loops 1, 3, 5, 7, and 9 face the extracellular side, and loops 2, 4, 6, and 10 face the cytoplasmic side. A re‐entrant loop‐like structure between TM8 and TM9 may protrude into the membrane. Our results support the topography of 11 transmembrane domains with an extracellular C terminus and an intracellular N terminus of NaDC3, and for the first time provide experimental evidence for a novel topological model for NaDC3—Bai, X.‐Y., Chen, X., Sun, A.‐Q., Feng, Z., Hou, K., Fu, B. Membrane topology structure of human high‐affinity, sodium‐dependent dicarboxylate transporter. FASEB J. 21, 2409–2417 (2007)

High concentrations of uric acid inhibit angiogenesis via regulation of the Krüppel-like factor 2-vascular endothelial growth factor-A axis by miR-92a.

BACKGROUND Angiogenesis is a critical component of many pathological conditions, and microRNAs (miRNAs) are indispensable in angiogenesis. It is unclear whether miRNAs regulate angiogenesis in the presence of high concentrations of uric acid (HUA), and the underlying mechanisms remain unknown. METHODSANDRESULTS It was found that HUA inhibited the angiogenic ability of endothelial cells. miRNA expression profiling was conducted using microarray assays in HUA-stimulated endothelial cells. Eighteen differentially expressed miRNAs were subjected to bioinformatic analyses. The results indicated that miR-92a was negatively regulated and was closely related to angiogenesis. Furthermore, the effects of miR-92a on HUA-stimulated endothelial cell angiogenesis and the underlying mechanisms were investigated in dual-luciferase reporter assays, electrophoretic mobility shift assays, immunoblot assays, and tube formation assays. It was determined that Krüppel-like factor 2 (KLF2) is a target gene of miR-92a, and KLF2 binds the vascular endothelial growth factor-A (VEGFA) promoter to inhibit its expression. miR-92a and VEGFA overexpression or KLF2 downregulation alleviates the HUA-mediated inhibition of angiogenesis in endothelial cells in vitro. CONCLUSIONS This study reported that there is a novel pathway regulating angiogenesis under HUA conditions. In the presence of HUA, miR-92a downregulation increased KLF2 expression, subsequently inhibiting VEGFA, which resulted in decreased angiogenesis. Thus, this study reports a possible mechanism for cardiovascular injury caused by hyperuricemia and suggests that the miR-92a-KLF2-VEGFA axis may be a target for hyperuricemia treatment.

Dysmorphic erythrocytes are superior to hematuria for indicating non-diabetic renal disease in type 2 diabetics

There are sparse and limited studies on erythrocyte morphology in renal biopsy identifying nephropathic patients among type 2 diabetics. The present study sought to clarify the predictive value of dysmorphic erythrocytes in type 2 diabetics with non‐diabetic renal disease and influences on hematuria.

Expression of EGFP/SDCT1 fusion protein, subcellular localization signal analysis, tissue distribution and electrophysiological function study.

Full-length cDNA gene of sodium-dependent dicarboxylate co-transporter protein 1 (SDCT1) is cloned from normal human kidney tissue and inserted into EGFP (enhanced green fluorescent protein) expression vector along with N-terminal and C-terminal truncated SDCT1 genes, so to construct the eukaryotic expression vectors of EGFP/SDCT1 fusion proteins, which are transfected into human renal tubular epithelial cells (HKC). Subcellular localizations of these fusion proteins are observed by laser confocal microscope to determine the localization signal of the SDCT1 protein. Duplex PCR analysis validates that the fusion protein genes have been integrated into the genome of HKC. Western blot indicates that the fusion proteins have been expressed in HKC. Confocal microscopy analysis shows that human SDCT1 predominantly locates on the plasma membrane, which is consistent with the results predicted by bioinformatics approach; in HKC transfected with N-terminal truncated SDCT1 gene, the green fluorescence is mainly distributed on the plasma membrane; in HKC transfected with C-terminal truncated SDCT1 gene, the green fluorescence is mainly distributed in the cytoplasm. EGFP/SDCT1 mRNAs obtained by in vitro transcription are microinjected into Xenopus laevis oocytes for expression and the trans-membrane currents are measured by using two-microelectrode volt-age-clamp technique. Na+ inward currents are present on cellular membrane of the injected oocytes. Immunohistochemical staining shows that human SDCT1 proteins are expressed on lumen membrane of the renal proximal tubule, but are negative in distal tubule, collecting duct, renal interstitium and glomerulus. The above-mentioned studies suggest that human SDCT1 protein is located on the lumen membrane of the renal proximal tubule, the C-terminal sequence of the SDCT1 is required for delivery and targeting localization, and the plasma membrane localization signal of the SDCT1 protein maybe locate in the C-terminal sequence.