I am a Xue-Yuan Bai, a professer of Department of Nephrology, Chinese PLA General hospital, mainly engaged in the study of kidney diseases and immune-associated deseases.
Peripheral blood mononuclear cells (PBMCs) were isolated from a healthy male individual. PBMCs we... more Peripheral blood mononuclear cells (PBMCs) were isolated from a healthy male individual. PBMCs were reprogrammed into human induced pluripotent stem cells (iPSCs) using plasmids carrying the following reprogramming factors: NANOG, LIN28, OCT4, SOX2, c-MYC, and KLF4. This cell line expressed pluripotency markers, exhibited the normal male karyotype (46, XY), and had the potential to differentiate into 3 germ layers, as confirmed by techniques such as flow cytometry. The iPSC obtained from a healthy individual can be used for organoids to reveal developmental mechanism of tissue and organ regeneration, stem cell therapy and drug screening.
Background. Heterozygous GANAB mutations that can cause autosomal dominant polycystic kidney dise... more Background. Heterozygous GANAB mutations that can cause autosomal dominant polycystic kidney disease (ADPKD) and polycystic liver disease (PLD) have been described previously, but their roles in ADPKD and PLD are largely unknown. With the increase in polycystic kidney disease caused by GANAB gene mutations in recent years, a suitable animal model is still needed to further explore the pathogenic role of this gene. Methods. To construct a mouse model of Ganab gene deletion, we analyzed the Ganab gene structure and designed two CRISPR-/Cas9-based targeting strategies. The Cas9/sgRNA we constructed was microinjected into fertilized mouse eggs to obtain chimeric F0 mice. Mice with stable genotypes were selected from offspring born after mating F0 mice with wild-type mice. Results. We found that homozygous mutation of the Ganab gene in C57BL/6 mice resulted in early embryonic lethality, and there were no cysts in the kidneys or livers of Ganab+/- mice. Additionally, Ganab protein express...
Previous studies of Jak–STAT inhibitors have shown promise in treating kidney diseases. The activ... more Previous studies of Jak–STAT inhibitors have shown promise in treating kidney diseases. The activation of Jak–STAT components is important in cell fate determination in many cell types, including bone marrow–derived cells, which are important contributors in renal interstitial fibrosis. In this study, we tested the effect of a new STAT3 inhibitor, BP-1-102, on monocyte-to-fibrocyte transition and the progression of renal interstitial fibrosis. We tested the effect of BP-1-102 in a mouse model of unilateral ureteral obstruction in vivo and IL-33-treated bone marrow–derived monocytes in vitro. BP-1-102 treatment alleviated renal interstitial fibrosis, reduced collagen deposition and extracellular matrix protein production, inhibited inflammatory cell infiltration, suppressed the percentage of CD45+ PDGFRβ+, CD45+ CD34− Col I+ and CD45+ CD11b+ Col I+ cells within the obstructed kidney and reduced the mRNA levels of the proinflammatory and profibrotic cytokines IL-1β, TGF-β, TNF-α, ICAM...
We established a young (Y)-old (O) rat kidney transplantation model. With this model, we detected... more We established a young (Y)-old (O) rat kidney transplantation model. With this model, we detected no age-related differences in renal structure between Y→Y and Y→O kidneys or O→O and O→Y kidneys. However, we did detect differences in levels of the senescence markers β-gal and p16 as well as the inflammatory cytokines TNF-α and IL-1β. Using proteomics analysis we detected 66 proteins associated with suppression of aging and 73 proteins associated with enhancement of aging. After construction of a protein-protein interaction network, a total of 73 nodes and 99 edges were analyzed using MCODE, and three significant modules were selected. GO and KEGG analyses showed that these proteins were mainly located in mitochondria and were largely related to oxidative stress. Among them, SOD1 expression was lower in Y→O than Y→Y kidneys and higher in O→Y than O→O kidneys. Acetylated (Ac)-NF-κB showed the opposite expression profile. In addition, SOD1 expression was higher in primary tubular epith...
Mesangial proliferative glomerulonephritis (MsPGN) is an inflammatory disease, but both the natur... more Mesangial proliferative glomerulonephritis (MsPGN) is an inflammatory disease, but both the nature of disease progression and its regulation remain unclear. In the present study, we monitored the course of anti-Thy1 nephritis from days 1 to 5 and established gene expression profiles at each time point using microarrays to explore the development of inflammation. According to the gene expression profiles, macrophage infiltration (triggered by CCL2 activation) was evident on day 1 and enhanced inflammation over the next few days. We screened for genes with expression levels similar to CCL2 and found that the upregulation of the circadian gene albumin D-site-binding protein (DBP) was involved in CCL2 activation in mesangial cells. More importantly, CCL2 expression showed oscillatory changes similar to DBP, and DBP induced peak CCL2 expression at 16:00 a clock on day 1 in the anti-Thy1 nephritis model. We knocked down DBP through transfection with a small interfering RNA (siRNA) and use...
The mechanisms of kidney aging are not yet clear. Studies have shown that immunological inflammat... more The mechanisms of kidney aging are not yet clear. Studies have shown that immunological inflammation is related to kidney aging. Toll-like receptors (TLRs) are one of the receptor types of the body's innate immune system. The function of the TLR system and the mechanisms by which it functions in renal aging remain unclear. In the present study, we, for the first time, systematically investigated the role of the TLR system and the inflammation responses activated by TLRs during kidney aging. We used western blot and immunohistochemistry to systematically analyze the changes in the expression and activation of the endogenous TLR ligands HSP70 and HMGB1, the TLRs (TLR1-TLR11), their downstream signaling pathway molecules MyD88 and Phospho-IRF-3, and the NF-κB signaling pathway molecules Phospho-IKKβ, Phospho-IκBα (NF-κB inhibition factor α), NF-κBp65, and Phospho-NF-κBp65 (activated NF-κB p65) in the kidneys of 3 months old (youth group), 12 months old (middle age group), and 24 mo...
The journals of gerontology. Series A, Biological sciences and medical sciences, Jan 9, 2014
High-affinity sodium-dependent dicarboxylate cotransporter 3 (NaDC3) is a key metabolism-regulati... more High-affinity sodium-dependent dicarboxylate cotransporter 3 (NaDC3) is a key metabolism-regulating membrane protein responsible for transport of Krebs cycle intermediates. NaDC3 is upregulated as organs age, but knowledge regarding the underlying mechanisms by which NaDC3 modulates mammalian aging is limited. In this study, we showed that NaDC3 overexpression accelerated cellular senescence in young human diploid cells (MRC-5 and WI-38) and primary renal tubular cells, leading to cell cycle arrest in G1 phase and increased expression of senescent biomarkers, senescence-associated β-galactosidase and p16. Intracellular levels of reactive oxygen species, 8-hydroxy-2'-deoxyguanosine, malondialdehyde, and carbonyl were significantly enhanced, and activities of respiratory complexes I and III and ATP level were significantly decreased in NaDC3-infected cells. Stressful premature senescent phenotypes induced by NaDC3 were markedly ameliorated via treatment with the antioxidants Tiron...
International Journal of Biological Sciences, 2015
PKD1 and PKD2 mutations could lead to autosomal dominant polycystic kidney disease (ADPKD), which... more PKD1 and PKD2 mutations could lead to autosomal dominant polycystic kidney disease (ADPKD), which afflicts millions of people worldwide. Due to the marked differences in the lifespan, size, anatomy, and physiology from humans, rodent ADPKD models cannot fully mimic the disease. To obtain a large animal model that recapitulates the disease, we constructed a mini-pig model by mono-allelic knockout (KO) of PKD1 using zinc finger nuclease. The mono-allelic KO pigs had lower PKD1 expression than their wild-type littermates at both the transcriptional and translational levels. After approximately six months, renal cysts appeared and grew progressively in the KO pigs. Histological analysis showed that renal cysts were scatteredly distributed in the mutant pig kidneys and were lined by either cuboidal or flattened epithelial cells. Contrast-enhanced computed tomography confirmed that all of the mutant pigs had renal and hepatic cysts, when they were 11-month-old. Immunohistochemical analysis revealed that most of the cysts were derived from the proximal tubules and collecting ducts. Therefore, the PKD1 mono-allelic knockout is sufficient to trigger renal cystogenesis, and this pig model may provide a platform for future study of renal cyst formation.
Autosomal dominant polycystic kidney disease (ADPKD) is a common human genetic disease, affecting... more Autosomal dominant polycystic kidney disease (ADPKD) is a common human genetic disease, affecting millions of people worldwide. The progressive growth of cysts in kidneys eventually leads to renal failure in 50 % of patients, and there is currently no effective treatment. Various murine models have been studied to elucidate the disease mechanisms, and much information has been acquired. However, the course of the disease cannot be fully recapitulated using these models. The pig is a suitable model for biomedical research, and pig PKD2 has high similarity to the human ortholog at the molecular level. Here, a mini-pig PKD2 transgenic model was generated, driven by a ubiquitous cytomegalovirus enhancer/promoter. Using somatic cell nuclear transfer, four transgenic pigs with approximately 10 insertion events each were generated. Quantitative real-time PCR and western blotting showed that PKD2 was more highly expressed in transgenic pigs than in wild-type counterparts. Because of the chronic nature of ADPKD, blood urea nitrogen and serum creatinine levels were continuously measured to assess the pig kidney function. The transgenic pigs continue to show no significant alteration in kidney function; it is estimated that 1-2 more years may be required for manifestation of renal cystogenesis in these pigs.
After several decades of research, autosomal dominant polycystic kidney disease (ADPKD) is still ... more After several decades of research, autosomal dominant polycystic kidney disease (ADPKD) is still incurable and imposes enormous physical, psychological, and economic burdens on patients and their families. Murine models of ADPKD represent invaluable tools for studying this disease. These murine forms of ADPKD can arise spontaneously, or they can be induced via chemical or genetic manipulations. Although these models have improved our understanding of the etiology and pathogenesis of ADPKD, they have not led to effective treatment strategies. The mini-pig represents an effective biomedical model for studying human diseases, as the pig's human-like physiological processes help to understand disease mechanisms and to develop novel therapies. Here, we tried to generate a transgenic model of ADPKD in pigs by overexpressing c-Myc in kidney tissue. Western-blot analysis showed that c-Myc was overexpressed in the kidney, brain, heart, and liver of transgenic pigs. Immunohistochemical staining of kidney tissue showed that exogenous c-Myc predominantly localized to renal tubules. Slightly elevated blood urea nitrogen levels were observed in transgenic pigs 1 month after birth, but no obvious abnormalities were detected after that time. In the future, we plan to subject this model to renal injury in an effort to promote ADPKD progression.
Adipose-derived stem cells (ASCs) can alleviate acute kidney injury and promote kidney cell regen... more Adipose-derived stem cells (ASCs) can alleviate acute kidney injury and promote kidney cell regeneration and repair. To investigate the role of ASCs in diabetic nephropathy (DN), Sprague-Dawley rats were made diabetic by intraperitoneal injection of streptozotocin (STZ) after uninephrectomy. After 12 weeks, proteinuria was well established. Five times of 5 • 10 6 human ASCs repeatedly injected through a tail vein at 4 weekly intervals. A reduction in proteinuria was not observed in diabetic rats until 24 weeks. However, urinary protein excretion was significantly suppressed at 28 weeks and persisted up to 32 weeks after STZ treatment. ASC treatment significantly attenuated glomerulus hypertrophy and tubular interstitial injury, and led to the downregulation of WT-1 and synaptopodin expression. CFSE labeled ASCs were injected into DN rats via the tail vein. Within 24 h after injection, the cells were detected in lung, spleen, and peritubular regions, but rarely in pancreas. Human Alu gene expression was detected in lung and spleen up to 4 weeks after ASCs injection. ASC treatment did not improve hyperglycemia or pancreatic damage. In vitro, recombinant human glial cell line-derived neurotrophic factor (GDNF) prevented podocyte injury by high glucose similarly to ASC-conditioned medium. After blocking GDNF in ASC-CM with neutralizing antibody, the therapeutic effect of ASC-CM was significantly decreased. ASCs cocultured with podocytes restored the downregulation of synaptopodin expression, which was weakened by GDNF-RNA interfering. These findings indicate that repeated intravenous ASC can reduce diabetic kidney damage in rats even at the progressive stage, and promote podocyte recovery via GDNF secretion.
Anti-Thy1 nephritis is a well-established experimental mesangial proliferative nephritis model. E... more Anti-Thy1 nephritis is a well-established experimental mesangial proliferative nephritis model. Exploring the molecular mechanisms of pathophysiology in anti-Thy1 nephritis may elucidate the pathogeneses of mesangial proliferation. We examined the roles and acting mechanisms of differentially expressed proteins (DEPs) by bioinformatics analysis of glomeruli proteomic profiles during the course of anti-Thy1 nephritis. In total, 108 DEPs were found by two-dimensional fluorescence difference gel electrophoresis (2D-DIGE), and 40 DEPs were identified by matrix-assisted laser desorption ionization/time of flight and liquid chromatography-MS. DEPs were classified into five clusters (Clusters 1-5), according to their expression trends using Cluster 3.0 software, involved in regulating biological processes such as the stress response, cell proliferation, apoptosis, energy metabolism, transport, and the actin cytoskeleton. The expression patterns of ten DEPs, distributed across five clusters, including AKR1A1, AGAT, ATP6V1B2, HIBADH, MDH1, MPST, NIT2, PRDX6, PSMB7, and TPI1, were validated by Western blotting. Based on Western blotting and immunohistochemistry, we also found that the DEP FHL2, which was primarily expressed in the mesangial region, was downregulated on days 3 and 5, and up-regulated on day 10. In vitro, we found that FHL2 overexpression induced mesangial cell proliferation by increasing the number of Sphase cells and decreasing G2/M-phase cells, whereas inhibiting FHL2 had the opposite effect. This study explored novel DEPs and their expression patterns during anti-Thy1 nephritis, and elucidated FHL2's effect on mesangial cell proliferation. These results will contribute to our understanding of the pathogenesis of mesangial proliferation.
Objective Glomerular filtration rate (GFR) is an important indicator of renal function. This meta... more Objective Glomerular filtration rate (GFR) is an important indicator of renal function. This meta-analysis aimed to evaluate the diagnostic value of serum cystatin C (CysC) and serum creatinine (SCr) for estimating GFR in patients with chronic kidney disease. Methods Google Scholar, PubMed®, Cochrane Library and China National Knowledge Infrastructure databases were searched, to identify randomized controlled trials that determined the diagnostic value of CysC and SCr, for estimating GFR in patients with chronic kidney disease. Results The inclusion criteria were met by 17 studies (total number of patients with chronic kidney disease, 2521). Meta-analysis showed that when the diagnostic cut-off value of GFR was 80–90 ml/min/1.73 m2, the heterogeneity was modest for CysC ( I2 = 48%, summary sensitivity [SEN] = 0.803, summary specificity [SPE] = 0.821), but there was no heterogeneity for SCr ( I2 = 0.0%, SEN = 0.697, SPE = 0.787). Meta-analysis of the studies demonstrated a significan...
could extend the G0/G1 phase and block cell proliferation in RMCs. Dual-luciferase assay results ... more could extend the G0/G1 phase and block cell proliferation in RMCs. Dual-luciferase assay results showed that there were binding sites of miR-34a at 3′-UTR of plateletderived growth factor receptor-β (PDGFR-β). MiR-34a can inhibit PDGFR-β protein expression at a post-transcriptional level, suppress Ras/MAPK signaling pathways, and down-regulate expression of cell cycle proteins at the G0/ G1 phase, such as cyclin D1, CDK4/CDK6. In addition, miR-34a may also inhibit RMC proliferation by directly targeting cyclin E and CDK2. MiR-34a inhibits exogenous stimuli-induced proliferation of mesangial cells. Expression levels of phospho-PDGFR-β and phospho-MEK1 (an important downstream molecule in PDGFR-β-induced signaling pathway) were significantly increased in the anti-Thy-1 nephritis rat model. These results suggest that miR-34a may regulate RMC proliferation by directly inhibiting expressions of PDGFR-β, MEK1, and cell cycle proteins, cyclin E and CDK2.
Immunofluorescence of frozen tissue sections (IF-F) is a classic technique for renal immunopathol... more Immunofluorescence of frozen tissue sections (IF-F) is a classic technique for renal immunopathologic examination. However, it has certain disadvantages, such as diffuse antigen distribution and few or even no glomeruli in the section. We developed a new technique of immunofluorescence staining using dual microwave retrieval in paraffinembedded renal tissue sections (IF-DMP) and compared IF-DMP with IF-F in 406 renal biopsy samples. IF-DMP detected significantly more glomeruli than did IF-F (P < .001). There was no significant difference for the specificity and sensitivity in the detection of immunoglobulins, complements, k, and l between IF-F and IF-DMP. Concordant observations were 98% for all immunofluorescence, complements, k, and l staining and 100% for immunoglobulin staining. Both techniques were completely accurate in confirming diagnoses of various glomerular diseases. IF-DMP provided clearer images of tissue structure and more precise localization of antigens, and it is a suitable alternative for traditional IF-F in clinical renal immunopathologic diagnosis.
Background and aims: It has been considered that the functional decline of renal vasoconstriction... more Background and aims: It has been considered that the functional decline of renal vasoconstriction during senescence is associated with an alteration in renal α 1-adrenergic receptor (α 1-AR) expression. While alterations in renal angiotensin II receptor (ATR) expression was considered to have an effect on renal structure and function, until now little information has been available concerning α 1-AR and ATR expression variations over the entire aging continuum. The present study was undertaken to examine the expression levels of α 1-AR and ATR subtypes in renal tissue during the spectrum running from young adulthood, to middle age, to the presenium, and to the senium. Methods: Semiquantitative Reverse Transcription Polymerase Chain Reaction (RT-PCR) and Western Blot were used to quantify the messenger RNA (mRNA) and protein levels of α 1-AR and ATR subtypes in renal tissue in 3-monthold (young adult), 12-month-old (middle age), 18-monthold (presenium) and 24-month-old (senium) Wistar rats. Results: α 1A-AR expression decreased gradually with aging: it was decreased during middle age, the presenium and the senium, compared, respectively, with young adult values (p<0.01), and there was a significant decline both in the presenium with respect to middle age and in the senium with respect to the presenium. α 1B-AR and α 1D-AR expression were unmodified during senescence. AT 1 R expression was unaffected by aging during young adulthood and middle age, but exhibited a remarkable downregulation in the presenium and senium periods (p<0.01). AT 2 R expression was markedly increased in the senium (p<0.01). Conclusions: These results suggest that there are considerable variations in the expression levels of renal α 1-AR and ATR subtypes during aging. α 1A-AR expression downregulation may account for the reduced reactivity of renal α 1-AR to vasoconstrictors and to renal function decline in the senium. Both the downregulation of AT 1 R and the upregulation of AT 2 R may be influential in maintaining normal physiological renal function during aging.
The mechanisms of diabetic renal injury remain unclear. Recent studies have shown that immunologi... more The mechanisms of diabetic renal injury remain unclear. Recent studies have shown that immunological and inflammatory elements play important roles in the initiation and development of diabetic nephropathy (DN). Toll-like receptors (TLRs) comprise a superfamily of innate immune system receptors. The roles and mechanisms of TLRs in the pathogenesis of diabetic renal lesions are mostly unknown. Compared with rodents, miniature pigs are more similar to humans with respect to metabolism, kidney structure, and immune system, and therefore represent an ideal large-animal model for DN mechanistic studies. A diabetes model was established by feeding miniature pigs with high-sugar and high-fat diets. Functional and pathological markers, expression and activation of endogenous TLR ligands [HSP70 (heat shock protein 70) and HMGB1], TLR1 to TLR11 and their downstream signaling pathway molecules (MyD88, IRAK-1, and IRF-3), nuclear factor κB (NF-κB) signaling pathway molecules (IKKβ, IκBα, and NF...
Sirtuin 3 (SIRT3) is one of the seven mammalian sirtuins, which are homologs of the yeast Sir2 ge... more Sirtuin 3 (SIRT3) is one of the seven mammalian sirtuins, which are homologs of the yeast Sir2 gene. SIRT3 is the only sirtuin reported to be associated with human life span. Many recent studies have indicated that SIRT3 levels are elevated by exercise and caloric restriction, but whether SIRT3 influences cell senescence under stressed conditions in human diploid fibroblasts has not been established. Our data showed that expression of AGE
Peripheral blood mononuclear cells (PBMCs) were isolated from a healthy male individual. PBMCs we... more Peripheral blood mononuclear cells (PBMCs) were isolated from a healthy male individual. PBMCs were reprogrammed into human induced pluripotent stem cells (iPSCs) using plasmids carrying the following reprogramming factors: NANOG, LIN28, OCT4, SOX2, c-MYC, and KLF4. This cell line expressed pluripotency markers, exhibited the normal male karyotype (46, XY), and had the potential to differentiate into 3 germ layers, as confirmed by techniques such as flow cytometry. The iPSC obtained from a healthy individual can be used for organoids to reveal developmental mechanism of tissue and organ regeneration, stem cell therapy and drug screening.
Background. Heterozygous GANAB mutations that can cause autosomal dominant polycystic kidney dise... more Background. Heterozygous GANAB mutations that can cause autosomal dominant polycystic kidney disease (ADPKD) and polycystic liver disease (PLD) have been described previously, but their roles in ADPKD and PLD are largely unknown. With the increase in polycystic kidney disease caused by GANAB gene mutations in recent years, a suitable animal model is still needed to further explore the pathogenic role of this gene. Methods. To construct a mouse model of Ganab gene deletion, we analyzed the Ganab gene structure and designed two CRISPR-/Cas9-based targeting strategies. The Cas9/sgRNA we constructed was microinjected into fertilized mouse eggs to obtain chimeric F0 mice. Mice with stable genotypes were selected from offspring born after mating F0 mice with wild-type mice. Results. We found that homozygous mutation of the Ganab gene in C57BL/6 mice resulted in early embryonic lethality, and there were no cysts in the kidneys or livers of Ganab+/- mice. Additionally, Ganab protein express...
Previous studies of Jak–STAT inhibitors have shown promise in treating kidney diseases. The activ... more Previous studies of Jak–STAT inhibitors have shown promise in treating kidney diseases. The activation of Jak–STAT components is important in cell fate determination in many cell types, including bone marrow–derived cells, which are important contributors in renal interstitial fibrosis. In this study, we tested the effect of a new STAT3 inhibitor, BP-1-102, on monocyte-to-fibrocyte transition and the progression of renal interstitial fibrosis. We tested the effect of BP-1-102 in a mouse model of unilateral ureteral obstruction in vivo and IL-33-treated bone marrow–derived monocytes in vitro. BP-1-102 treatment alleviated renal interstitial fibrosis, reduced collagen deposition and extracellular matrix protein production, inhibited inflammatory cell infiltration, suppressed the percentage of CD45+ PDGFRβ+, CD45+ CD34− Col I+ and CD45+ CD11b+ Col I+ cells within the obstructed kidney and reduced the mRNA levels of the proinflammatory and profibrotic cytokines IL-1β, TGF-β, TNF-α, ICAM...
We established a young (Y)-old (O) rat kidney transplantation model. With this model, we detected... more We established a young (Y)-old (O) rat kidney transplantation model. With this model, we detected no age-related differences in renal structure between Y→Y and Y→O kidneys or O→O and O→Y kidneys. However, we did detect differences in levels of the senescence markers β-gal and p16 as well as the inflammatory cytokines TNF-α and IL-1β. Using proteomics analysis we detected 66 proteins associated with suppression of aging and 73 proteins associated with enhancement of aging. After construction of a protein-protein interaction network, a total of 73 nodes and 99 edges were analyzed using MCODE, and three significant modules were selected. GO and KEGG analyses showed that these proteins were mainly located in mitochondria and were largely related to oxidative stress. Among them, SOD1 expression was lower in Y→O than Y→Y kidneys and higher in O→Y than O→O kidneys. Acetylated (Ac)-NF-κB showed the opposite expression profile. In addition, SOD1 expression was higher in primary tubular epith...
Mesangial proliferative glomerulonephritis (MsPGN) is an inflammatory disease, but both the natur... more Mesangial proliferative glomerulonephritis (MsPGN) is an inflammatory disease, but both the nature of disease progression and its regulation remain unclear. In the present study, we monitored the course of anti-Thy1 nephritis from days 1 to 5 and established gene expression profiles at each time point using microarrays to explore the development of inflammation. According to the gene expression profiles, macrophage infiltration (triggered by CCL2 activation) was evident on day 1 and enhanced inflammation over the next few days. We screened for genes with expression levels similar to CCL2 and found that the upregulation of the circadian gene albumin D-site-binding protein (DBP) was involved in CCL2 activation in mesangial cells. More importantly, CCL2 expression showed oscillatory changes similar to DBP, and DBP induced peak CCL2 expression at 16:00 a clock on day 1 in the anti-Thy1 nephritis model. We knocked down DBP through transfection with a small interfering RNA (siRNA) and use...
The mechanisms of kidney aging are not yet clear. Studies have shown that immunological inflammat... more The mechanisms of kidney aging are not yet clear. Studies have shown that immunological inflammation is related to kidney aging. Toll-like receptors (TLRs) are one of the receptor types of the body's innate immune system. The function of the TLR system and the mechanisms by which it functions in renal aging remain unclear. In the present study, we, for the first time, systematically investigated the role of the TLR system and the inflammation responses activated by TLRs during kidney aging. We used western blot and immunohistochemistry to systematically analyze the changes in the expression and activation of the endogenous TLR ligands HSP70 and HMGB1, the TLRs (TLR1-TLR11), their downstream signaling pathway molecules MyD88 and Phospho-IRF-3, and the NF-κB signaling pathway molecules Phospho-IKKβ, Phospho-IκBα (NF-κB inhibition factor α), NF-κBp65, and Phospho-NF-κBp65 (activated NF-κB p65) in the kidneys of 3 months old (youth group), 12 months old (middle age group), and 24 mo...
The journals of gerontology. Series A, Biological sciences and medical sciences, Jan 9, 2014
High-affinity sodium-dependent dicarboxylate cotransporter 3 (NaDC3) is a key metabolism-regulati... more High-affinity sodium-dependent dicarboxylate cotransporter 3 (NaDC3) is a key metabolism-regulating membrane protein responsible for transport of Krebs cycle intermediates. NaDC3 is upregulated as organs age, but knowledge regarding the underlying mechanisms by which NaDC3 modulates mammalian aging is limited. In this study, we showed that NaDC3 overexpression accelerated cellular senescence in young human diploid cells (MRC-5 and WI-38) and primary renal tubular cells, leading to cell cycle arrest in G1 phase and increased expression of senescent biomarkers, senescence-associated β-galactosidase and p16. Intracellular levels of reactive oxygen species, 8-hydroxy-2'-deoxyguanosine, malondialdehyde, and carbonyl were significantly enhanced, and activities of respiratory complexes I and III and ATP level were significantly decreased in NaDC3-infected cells. Stressful premature senescent phenotypes induced by NaDC3 were markedly ameliorated via treatment with the antioxidants Tiron...
International Journal of Biological Sciences, 2015
PKD1 and PKD2 mutations could lead to autosomal dominant polycystic kidney disease (ADPKD), which... more PKD1 and PKD2 mutations could lead to autosomal dominant polycystic kidney disease (ADPKD), which afflicts millions of people worldwide. Due to the marked differences in the lifespan, size, anatomy, and physiology from humans, rodent ADPKD models cannot fully mimic the disease. To obtain a large animal model that recapitulates the disease, we constructed a mini-pig model by mono-allelic knockout (KO) of PKD1 using zinc finger nuclease. The mono-allelic KO pigs had lower PKD1 expression than their wild-type littermates at both the transcriptional and translational levels. After approximately six months, renal cysts appeared and grew progressively in the KO pigs. Histological analysis showed that renal cysts were scatteredly distributed in the mutant pig kidneys and were lined by either cuboidal or flattened epithelial cells. Contrast-enhanced computed tomography confirmed that all of the mutant pigs had renal and hepatic cysts, when they were 11-month-old. Immunohistochemical analysis revealed that most of the cysts were derived from the proximal tubules and collecting ducts. Therefore, the PKD1 mono-allelic knockout is sufficient to trigger renal cystogenesis, and this pig model may provide a platform for future study of renal cyst formation.
Autosomal dominant polycystic kidney disease (ADPKD) is a common human genetic disease, affecting... more Autosomal dominant polycystic kidney disease (ADPKD) is a common human genetic disease, affecting millions of people worldwide. The progressive growth of cysts in kidneys eventually leads to renal failure in 50 % of patients, and there is currently no effective treatment. Various murine models have been studied to elucidate the disease mechanisms, and much information has been acquired. However, the course of the disease cannot be fully recapitulated using these models. The pig is a suitable model for biomedical research, and pig PKD2 has high similarity to the human ortholog at the molecular level. Here, a mini-pig PKD2 transgenic model was generated, driven by a ubiquitous cytomegalovirus enhancer/promoter. Using somatic cell nuclear transfer, four transgenic pigs with approximately 10 insertion events each were generated. Quantitative real-time PCR and western blotting showed that PKD2 was more highly expressed in transgenic pigs than in wild-type counterparts. Because of the chronic nature of ADPKD, blood urea nitrogen and serum creatinine levels were continuously measured to assess the pig kidney function. The transgenic pigs continue to show no significant alteration in kidney function; it is estimated that 1-2 more years may be required for manifestation of renal cystogenesis in these pigs.
After several decades of research, autosomal dominant polycystic kidney disease (ADPKD) is still ... more After several decades of research, autosomal dominant polycystic kidney disease (ADPKD) is still incurable and imposes enormous physical, psychological, and economic burdens on patients and their families. Murine models of ADPKD represent invaluable tools for studying this disease. These murine forms of ADPKD can arise spontaneously, or they can be induced via chemical or genetic manipulations. Although these models have improved our understanding of the etiology and pathogenesis of ADPKD, they have not led to effective treatment strategies. The mini-pig represents an effective biomedical model for studying human diseases, as the pig's human-like physiological processes help to understand disease mechanisms and to develop novel therapies. Here, we tried to generate a transgenic model of ADPKD in pigs by overexpressing c-Myc in kidney tissue. Western-blot analysis showed that c-Myc was overexpressed in the kidney, brain, heart, and liver of transgenic pigs. Immunohistochemical staining of kidney tissue showed that exogenous c-Myc predominantly localized to renal tubules. Slightly elevated blood urea nitrogen levels were observed in transgenic pigs 1 month after birth, but no obvious abnormalities were detected after that time. In the future, we plan to subject this model to renal injury in an effort to promote ADPKD progression.
Adipose-derived stem cells (ASCs) can alleviate acute kidney injury and promote kidney cell regen... more Adipose-derived stem cells (ASCs) can alleviate acute kidney injury and promote kidney cell regeneration and repair. To investigate the role of ASCs in diabetic nephropathy (DN), Sprague-Dawley rats were made diabetic by intraperitoneal injection of streptozotocin (STZ) after uninephrectomy. After 12 weeks, proteinuria was well established. Five times of 5 • 10 6 human ASCs repeatedly injected through a tail vein at 4 weekly intervals. A reduction in proteinuria was not observed in diabetic rats until 24 weeks. However, urinary protein excretion was significantly suppressed at 28 weeks and persisted up to 32 weeks after STZ treatment. ASC treatment significantly attenuated glomerulus hypertrophy and tubular interstitial injury, and led to the downregulation of WT-1 and synaptopodin expression. CFSE labeled ASCs were injected into DN rats via the tail vein. Within 24 h after injection, the cells were detected in lung, spleen, and peritubular regions, but rarely in pancreas. Human Alu gene expression was detected in lung and spleen up to 4 weeks after ASCs injection. ASC treatment did not improve hyperglycemia or pancreatic damage. In vitro, recombinant human glial cell line-derived neurotrophic factor (GDNF) prevented podocyte injury by high glucose similarly to ASC-conditioned medium. After blocking GDNF in ASC-CM with neutralizing antibody, the therapeutic effect of ASC-CM was significantly decreased. ASCs cocultured with podocytes restored the downregulation of synaptopodin expression, which was weakened by GDNF-RNA interfering. These findings indicate that repeated intravenous ASC can reduce diabetic kidney damage in rats even at the progressive stage, and promote podocyte recovery via GDNF secretion.
Anti-Thy1 nephritis is a well-established experimental mesangial proliferative nephritis model. E... more Anti-Thy1 nephritis is a well-established experimental mesangial proliferative nephritis model. Exploring the molecular mechanisms of pathophysiology in anti-Thy1 nephritis may elucidate the pathogeneses of mesangial proliferation. We examined the roles and acting mechanisms of differentially expressed proteins (DEPs) by bioinformatics analysis of glomeruli proteomic profiles during the course of anti-Thy1 nephritis. In total, 108 DEPs were found by two-dimensional fluorescence difference gel electrophoresis (2D-DIGE), and 40 DEPs were identified by matrix-assisted laser desorption ionization/time of flight and liquid chromatography-MS. DEPs were classified into five clusters (Clusters 1-5), according to their expression trends using Cluster 3.0 software, involved in regulating biological processes such as the stress response, cell proliferation, apoptosis, energy metabolism, transport, and the actin cytoskeleton. The expression patterns of ten DEPs, distributed across five clusters, including AKR1A1, AGAT, ATP6V1B2, HIBADH, MDH1, MPST, NIT2, PRDX6, PSMB7, and TPI1, were validated by Western blotting. Based on Western blotting and immunohistochemistry, we also found that the DEP FHL2, which was primarily expressed in the mesangial region, was downregulated on days 3 and 5, and up-regulated on day 10. In vitro, we found that FHL2 overexpression induced mesangial cell proliferation by increasing the number of Sphase cells and decreasing G2/M-phase cells, whereas inhibiting FHL2 had the opposite effect. This study explored novel DEPs and their expression patterns during anti-Thy1 nephritis, and elucidated FHL2's effect on mesangial cell proliferation. These results will contribute to our understanding of the pathogenesis of mesangial proliferation.
Objective Glomerular filtration rate (GFR) is an important indicator of renal function. This meta... more Objective Glomerular filtration rate (GFR) is an important indicator of renal function. This meta-analysis aimed to evaluate the diagnostic value of serum cystatin C (CysC) and serum creatinine (SCr) for estimating GFR in patients with chronic kidney disease. Methods Google Scholar, PubMed®, Cochrane Library and China National Knowledge Infrastructure databases were searched, to identify randomized controlled trials that determined the diagnostic value of CysC and SCr, for estimating GFR in patients with chronic kidney disease. Results The inclusion criteria were met by 17 studies (total number of patients with chronic kidney disease, 2521). Meta-analysis showed that when the diagnostic cut-off value of GFR was 80–90 ml/min/1.73 m2, the heterogeneity was modest for CysC ( I2 = 48%, summary sensitivity [SEN] = 0.803, summary specificity [SPE] = 0.821), but there was no heterogeneity for SCr ( I2 = 0.0%, SEN = 0.697, SPE = 0.787). Meta-analysis of the studies demonstrated a significan...
could extend the G0/G1 phase and block cell proliferation in RMCs. Dual-luciferase assay results ... more could extend the G0/G1 phase and block cell proliferation in RMCs. Dual-luciferase assay results showed that there were binding sites of miR-34a at 3′-UTR of plateletderived growth factor receptor-β (PDGFR-β). MiR-34a can inhibit PDGFR-β protein expression at a post-transcriptional level, suppress Ras/MAPK signaling pathways, and down-regulate expression of cell cycle proteins at the G0/ G1 phase, such as cyclin D1, CDK4/CDK6. In addition, miR-34a may also inhibit RMC proliferation by directly targeting cyclin E and CDK2. MiR-34a inhibits exogenous stimuli-induced proliferation of mesangial cells. Expression levels of phospho-PDGFR-β and phospho-MEK1 (an important downstream molecule in PDGFR-β-induced signaling pathway) were significantly increased in the anti-Thy-1 nephritis rat model. These results suggest that miR-34a may regulate RMC proliferation by directly inhibiting expressions of PDGFR-β, MEK1, and cell cycle proteins, cyclin E and CDK2.
Immunofluorescence of frozen tissue sections (IF-F) is a classic technique for renal immunopathol... more Immunofluorescence of frozen tissue sections (IF-F) is a classic technique for renal immunopathologic examination. However, it has certain disadvantages, such as diffuse antigen distribution and few or even no glomeruli in the section. We developed a new technique of immunofluorescence staining using dual microwave retrieval in paraffinembedded renal tissue sections (IF-DMP) and compared IF-DMP with IF-F in 406 renal biopsy samples. IF-DMP detected significantly more glomeruli than did IF-F (P < .001). There was no significant difference for the specificity and sensitivity in the detection of immunoglobulins, complements, k, and l between IF-F and IF-DMP. Concordant observations were 98% for all immunofluorescence, complements, k, and l staining and 100% for immunoglobulin staining. Both techniques were completely accurate in confirming diagnoses of various glomerular diseases. IF-DMP provided clearer images of tissue structure and more precise localization of antigens, and it is a suitable alternative for traditional IF-F in clinical renal immunopathologic diagnosis.
Background and aims: It has been considered that the functional decline of renal vasoconstriction... more Background and aims: It has been considered that the functional decline of renal vasoconstriction during senescence is associated with an alteration in renal α 1-adrenergic receptor (α 1-AR) expression. While alterations in renal angiotensin II receptor (ATR) expression was considered to have an effect on renal structure and function, until now little information has been available concerning α 1-AR and ATR expression variations over the entire aging continuum. The present study was undertaken to examine the expression levels of α 1-AR and ATR subtypes in renal tissue during the spectrum running from young adulthood, to middle age, to the presenium, and to the senium. Methods: Semiquantitative Reverse Transcription Polymerase Chain Reaction (RT-PCR) and Western Blot were used to quantify the messenger RNA (mRNA) and protein levels of α 1-AR and ATR subtypes in renal tissue in 3-monthold (young adult), 12-month-old (middle age), 18-monthold (presenium) and 24-month-old (senium) Wistar rats. Results: α 1A-AR expression decreased gradually with aging: it was decreased during middle age, the presenium and the senium, compared, respectively, with young adult values (p<0.01), and there was a significant decline both in the presenium with respect to middle age and in the senium with respect to the presenium. α 1B-AR and α 1D-AR expression were unmodified during senescence. AT 1 R expression was unaffected by aging during young adulthood and middle age, but exhibited a remarkable downregulation in the presenium and senium periods (p<0.01). AT 2 R expression was markedly increased in the senium (p<0.01). Conclusions: These results suggest that there are considerable variations in the expression levels of renal α 1-AR and ATR subtypes during aging. α 1A-AR expression downregulation may account for the reduced reactivity of renal α 1-AR to vasoconstrictors and to renal function decline in the senium. Both the downregulation of AT 1 R and the upregulation of AT 2 R may be influential in maintaining normal physiological renal function during aging.
The mechanisms of diabetic renal injury remain unclear. Recent studies have shown that immunologi... more The mechanisms of diabetic renal injury remain unclear. Recent studies have shown that immunological and inflammatory elements play important roles in the initiation and development of diabetic nephropathy (DN). Toll-like receptors (TLRs) comprise a superfamily of innate immune system receptors. The roles and mechanisms of TLRs in the pathogenesis of diabetic renal lesions are mostly unknown. Compared with rodents, miniature pigs are more similar to humans with respect to metabolism, kidney structure, and immune system, and therefore represent an ideal large-animal model for DN mechanistic studies. A diabetes model was established by feeding miniature pigs with high-sugar and high-fat diets. Functional and pathological markers, expression and activation of endogenous TLR ligands [HSP70 (heat shock protein 70) and HMGB1], TLR1 to TLR11 and their downstream signaling pathway molecules (MyD88, IRAK-1, and IRF-3), nuclear factor κB (NF-κB) signaling pathway molecules (IKKβ, IκBα, and NF...
Sirtuin 3 (SIRT3) is one of the seven mammalian sirtuins, which are homologs of the yeast Sir2 ge... more Sirtuin 3 (SIRT3) is one of the seven mammalian sirtuins, which are homologs of the yeast Sir2 gene. SIRT3 is the only sirtuin reported to be associated with human life span. Many recent studies have indicated that SIRT3 levels are elevated by exercise and caloric restriction, but whether SIRT3 influences cell senescence under stressed conditions in human diploid fibroblasts has not been established. Our data showed that expression of AGE
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