LncRNA RPL29P2 promotes peritoneal fibrosis and impairs peritoneal transport function via miR-1184 in peritoneal dialysis.

Peritoneal dialysis (PD), hemodialysis and kidney transplantation are the three therapies to treat uremia. However, PD is discontinued for peritoneal membrane fibrosis (PMF) and loss of peritoneal transport function (PTF) due to damage from high concentrations of glucose in PD fluids (PDFs). The mechanism behind PMF is unclear, and there are no available biomarkers for the evaluation of PMF and PTF. Using microarray screening, we found that a new long noncoding RNA (lncRNA), RPL29P2, was upregulated in the PM (peritoneal membrane) of long-term PD patients, and its expression level was correlated with PMF severity and the PTF loss. In vitro and rat model assays suggested that lncRNA RPL29P2 targets miR-1184 and induces the expression of collagen type I alpha 1 chain (COL1A1). Silencing RPL29P2 in the PD rat model might suppress the HG-induced phenotypic transition of Human peritoneal mesothelial cells (HPMCs), alleviate HG-induced fibrosis and prevent the loss of PTF. Overall, our findings revealed that lncRNA RPL29P2, which targets miR-1184 and collagen, may represent a useful marker and therapeutic target of PMF in PD patients.

Comparison of different peritoneal dialysis catheters on complication and catheter survival: A network meta-analysis of randomised controlled trials.

BACKGROUND: This network meta-analysis (NMA) aimed to compare the clinical advantage of four commonly used peritoneal dialysis catheters (PDCs) including the Swan neck segment with straight tip (Swan neck + S), Tenckhoff segment with straight tip (Tenckhoff + S), Swan neck segment with coiled tip (Swan neck + C) and Tenckhoff segment with coiled tip (Tenckhoff + C). METHODS: Randomised clinical trials were searched from PubMed, Embase, the Cochrane Register of clinical trials, China National Knowledge Infrastructure (CNKI) and ChinaInfo from their inception until July 31, 2022. Meta-analysis was performed using Stata 14.0 and RevMan 5.3.5 software to evaluate the four commonly used PDCs. RESULTS: Seventeen studies involved 1578 participants were included. NMA showed that compared with Swan neck + C, Swan neck + S significantly reduced catheter tip migration (OR 0.47 95% CI 0.22-0.99). Tenckhoff + S was more effective in reducing catheter dysfunction (OR 0.42, 95% CI 0.23-0.79), catheter tip migration with dysfunction (OR 0.19, 95% CI 0.05-0.78) and catheter removal (OR 0.56, 95% CI 0.34-0.93) which were consistent with the pairwise meta-analysis. According to the surface under the cumulative ranking curve, Swan neck + S emerged as the best PDC in the reduction of catheter tip migration (83.3%), followed by Tenckhoff + S (79.4%). Moreover, Tenckhoff + S (86.5%, 76.3%) and Swan neck + S (72.3, 86.9%) ranked as the first and second PDC for 1 and 2-year technique survival which was significantly higher than those of the other two PDCs. CONCLUSION: Our NMA showed Swan neck + S and Tenckhoff + S tended to be more efficacious than Swan neck + C and Tenckhoff + C in lowering the mechanical dysfunction and prolonging the technique survival, which may contribute to better clinical decisions. More randomised controlled trials with larger scales and higher quality are needed in order to obtain more credible evidence.

Efficacy and safety of tacrolimus-based treatment for non-rapidly progressive IgA nephropathy.

In this study, we aimed to evaluate the efficacy and safety of tacrolimus-based treatment for immunoglobulin A nephropathy (IgAN). We retrospectively reviewed 127 adult patients with primary IgAN with 24 h urine total protein quantity (24 h UTP) ≥ 1 g and serum creatinine ≤3 mg/dL. All patients were divided into tacrolimus (TAC) and control (non-TAC) groups according to the treatment strategy. Proteinuria remission, remission rate, and adverse events were compared between the two groups. Among the 127 patients, 61 received TAC-based treatment and 66 received non-TAC treatment. TAC group exhibited a more rapid decline in proteinuria than the non-TAC group at 3, 9, and 12 months (p = 0.049, 0.001, and 0.018, respectively). Remission rates at 1, 3, 6, 9, and 12 months were 41.0, 68.9, 80.3, 90.2, and 88.5%, respectively, in the TAC group. These rates were higher than those in the control group at 3, 9, and 12 months (p = 0.030, 0.008, and 0.026, respectively). Complete remission rates at 1, 3, 6, 9, and 12 months were 6.56, 19.7, 37.7, 54.1, and 62.3%, respectively, in the TAC group. These rates were higher than those in the control group at 9 and 12 months (p = 0.013 and 0.008, respectively). The estimated mean time to complete remission was significantly shorter in the TAC group than in the control group (p = 0.028). TAC did not increase the incidence of adverse events. In conclusion, TAC accelerated proteinuria remission in patients with non-rapidly progressive IgAN with no increased risk of adverse events. Further prospective randomized controlled trials are necessary to validate our findings.

Short-term effects of distance training on peritoneal dialysis-related complications during the transmission control of COVID-19 epidemic in China.

OBJECTIVE: To observe the effects of distance training on patients with continuous ambulatory peritoneal dialysis (CAPD) during the transmission control of the COVID-19 epidemic in China. MATERIALS AND METHODS: CAPD patients from Xijing Hospital received a traditional training before the transmission control of COVID-19 epidemic, while they received a distance training dominated by WeChat and telephone during the transmission control of COVID-19 epidemic in China. Incidence and cure rate of PD-related peritonitis and catheter-related non-infectious complications were compared. All patients were followed up for 30 days from the date of complication. RESULTS: PD-related peritonitis, catheter displacement, and catheter occlusion had no significant difference, and the cure rate of PD-related peritonitis, catheter displacement, and catheter occlusion also had no significant difference in two comparisons despite the cure rate of PD-related peritonitis being slightly higher before (90.9%) than during (80%) the transmission control of COVID-19 epidemic. CONCLUSION: Distance training mode had a similar effect compared to the traditional training mode in the prevention and treatment of PD-related peritonitis and catheter-related non-infectious complications. PRACTICE IMPLICATIONS: Distance training model is an effective training mode that can be implemented in a short time during the epidemic period of serious infectious diseases.

Risk Factors and Management of Catheter Malfunction During Urgent-Start Peritoneal Dialysis.

Background: Catheter malfunction is a common complication of peritoneal dialysis (PD). This study aimed to retrospectively analyze the risk factors and management of catheter malfunction in urgent-start PD. Methods: Patients who underwent urgent-start PD were divided into catheter-malfunction and control groups. Baseline demographic and laboratory data of the two groups were compared, and the risk factors for catheter malfunction were analyzed. Primary outcome measure was catheter survival, and the secondary outcomes were surgical complications and malfunction treatment. Results: Total of 700 patients was analyzed, among whom 143 (20.4%) experienced catheter malfunctions, specifically catheter migration (96, 67.1%), omental wrapping (36, 25.2%), and migration plus omental wrapping (11, 7.7%). Catheter survival time in the malfunction group (202.5 ± 479.4 days) was significantly shorter than that in the control group (1295.3 ± 637.0 days) (P < 0.001). Multivariate analysis revealed higher body mass index [hazard ratio (HR), 1.061; 95% confidence intervals (CI), 1.010-1.115; P = 0.018], lower surgeon count (HR, 1.083; 95% CI, 1.032-1.136; P = 0.001), and higher serum potassium (HR, 1.231; 95% CI, 1.041-1.494; P = 0.036) as independent risk factors for catheter malfunction, while older age (HR, 0.976, 95% CI, 0.962-0.991; P = 0.002) and colonic dialysis (HR, 0.384; 95% CI, 0.254-0.581; P < 0.001) as protective factors. Further subgroup analysis revealed a shorter catheter survival time in patients with younger age ( ≤ 40 years), higher serum potassium levels (≥5 mmol/L), while a longer catheter survival time in patients with colonic dialysis. PD tube and subcutaneous tunnel preservation was successful in 41 out of 44 patients with omental wrapping. All patients had good post-incision prognoses. Conclusions: Urgent-start PD is safe and effective for unplanned PD patients. Adequate pre-operative colonic dialysis and serum potassium level control are conducive in preventing catheter malfunction. Conservative treatment is effective in managing catheter migration alone, while preservation of the PD tube and the subcutaneous tunnel is effective for omental wrapping.

Safety and effectiveness of lanthanum carbonate for hyperphosphatemia in chronic kidney disease (CKD) patients: a meta-analysis.

OBJECTIVE: The aim of this study was to determine the efficacy and safety of lanthanum carbonate (LC) versus calcium salts, non-LC phosphate binders (PBs), sevelamer, or placebo in patients with chronic kidney disease (CKD). MATERIALS AND METHODS: A literature search on PubMed, Embase, and Cochrane Library databases was conducted up to 18 June 2021. Data acquisition and quality assessment were performed by two reviewers. Meta-analysis was performed to evaluate the serum biochemical parameters, adverse events, and patient-level outcomes of LC, non-LC PBs, and sevelamer for hyperphosphatemia in patients with CKD. Heterogeneity across studies was assessed utilizing the I2 statistic and Q-test, and a random effect model was selected to calculate the pooled effect size. RESULTS: A total of 26 randomized, controlled trials and 3 observational studies were included. Compared to the other groups, better control effect of serum phosphorus (RR = 2.68, p < 0.001), reduction in serum phosphorus (95%CI = -1.93, -0.99; p < 0.001), Ca × P (95%CI = -13.89, -2.99; p = 0.002), serum intact parathyroid hormone levels (95%CI = -181.17, -3.96, p = 0.041) were found in LC group. Besides, reduced risk of various adverse effects, such as hypotension, abdominal pain, diarrhea, dyspepsia, and a score of coronary artery calcification were identified with LC in comparison to calcium salt, non-LC PBs, or placebo group. Significantly lower risk in mortality with LC treatment vs. non-LC PBs was observed, while no significant difference was identified between LC and calcium salt groups. CONCLUSION: LC might be an alternative treatment for hyperphosphatemia in patients with CKD considering its comprehensive curative effect.

Expression of XBP1s in B lymphocytes is critical for pristane-induced lupus nephritis in mice.

B lymphocyte hyperactivity plays a pathogenic role in systemic lupus erythematosus (SLE), and spliced X box-binding protein 1 (XBP1s) has been implicated in B cell maturation and differentiation. We hypothesized that blockade of the XBP1s pathway inhibits the B cell hyperactivity underlying SLE and lupus nephritis (LN) development. In the present study, we systematically evaluated the changes in B cell activation induced by the Xbp1 splicing inhibitor STF083010 in a pristane-induced lupus mouse model. The lupus mouse model was successfully established, as indicated by the presence of LN with markedly increased urine protein levels, renal deposition of Ig, and mesangial cell proliferation. In lupus mice, B cell hyperactivity was confirmed by increased CD40 and B cell-activating factor levels. B cell activation and plasma cell overproduction were determined by increases in CD40-positive and CD138-positive cells in the spleens of lupus mice by flow cytometry and further confirmed by CD45R and Ig light chain staining in the splenic tissues of lupus mice. mRNA and protein expression of XBP1s in B cells was assessed by real-time PCR, Western blot analysis, and immunofluorescence analysis and was increased in lupus mice. In addition, almost all changes were reversed by STF083010 treatment. However, the expression of XBP1s in the kidneys did not change when mice were exposed to pristane and STF083010. Taken together, these findings suggest that expression of XBP1s in B cells plays key roles in SLE and LN development. Blockade of the XBP1s pathway may be a potential strategy for SLE and LN treatment.

Expression of XBP1s in peritoneal mesothelial cells is critical for inflammation-induced peritoneal fibrosis.

Intraperitoneal inflammation is the most important determinant of peritoneal fibrosis in patients with long-term peritoneal dialysis (PD). Spliced x-box binding protein-1 (XBP1s), a major proximal effector of unfolded protein response (UPR) signaling, plays an indispensable role in inflammation. Our study demonstrated that the inflammatory factor interleukin-1ß (IL-1ß) dose- and time-dependently induced XBP1s upregulation and interleukin-6 (IL-6) secretion, as well as the expression of the fibrotic marker fibronectin. However, these effects were prevented by the IRE1 endonuclease inhibitor STF083010 since it time-dependently reduced IL-1ß-induced Xbp1 mRNA splicing, XBP1s protein expression, inflammatory factor IL-6 secretion and the expression of the fibrotic marker fibronectin in human peritoneal mesothelial cells (HPMCs). The overexpression and knockdown of XBP1s in HPMCs had a similar effect on fibronectin expression. In a rat model of peritoneal inflammation, STF083010 significantly attenuated chlorhexidine digluconate-induced XBP1s and α-smooth muscle actin expression, as well as fibrotic tissue proliferation, in the peritoneum. Our results suggest that XBP1s is a strong pathogenic factor that mediates inflammation-induced peritoneal fibrosis in peritoneal dialysis.

The landscape and diagnostic potential of T and B cell repertoire in Immunoglobulin A Nephropathy.

Immunoglobulin A Nephropathy (IgAN) is the most common glomerulonephritis worldwide. The pathologic hallmark of IgAN is immune complex deposited in glomerular mesangium, which induces inflammation and affects the kidney's normal functions. The exact pathogenesis of IgAN, however, remains obscure. Further, in current clinical practice, the diagnosis relies on needle biopsy of renal tissue. Therefore, a non-invasive method for diagnosis and prognosis surveillance of the disease is highly desirable. To this end, we investigated the T cell receptor beta chain (TCRB) and immunoglobulin heavy chain (IGH) repertoire in circulating lymphocytes and compared them with kidney infiltrating lymphocytes using immune repertoire high throughput sequencing. We found that some features of TCRB and IGH in renal tissues were remarkably different from that in the blood, including decreased repertoire diversity, increased IgA and IgG frequency, and more antigen-experienced B cells. The complementarity-determining region 3 (CDR3) length of circulating TCRB and IGH in IgAN patients was significantly shorter than that in healthy controls, which is the result of both VDJ rearrangement and clonal selection. The IgA1 frequency in the blood of IgAN patients is significantly higher than that in other Nephropathy (NIgAN) patients and healthy control. Importantly we identified a set of TCRB and IGH clones, which can be used to distinguish IgAN from NIgAN and healthy controls with high accuracy. These results indicated that the TCRB and IGH repertoire can potentially serve as non-invasive biomarkers for the diagnosis of IgAN. The characteristics of the kidney infiltrating and circulating lymphocytes repertoires shed light on IgAN detection, treatment and surveillance.

The MicroRNA-199a/214 Cluster Targets E-Cadherin and Claudin-2 and Promotes High Glucose-Induced Peritoneal Fibrosis.

Serum response factor (SRF) was found to be involved in the phenotypic transition and fibrosis of the peritoneal membrane during treatment with peritoneal dialysis (PD), but the exact mechanism remains unclear. SRF regulates microRNAs (miRNAs) that contain the SRF-binding consensus (CArG) element in the promoter region. Therefore, we investigated whether the miR-199a/214 gene cluster, which contains a CArG element in its promoter, is directly regulated by SRF. High-glucose (HG) treatment significantly unregulated the expression of the miR-199a-5p/214-3p gene cluster in human peritoneal mesothelial cells (HPMCs). By chromatin immunoprecipitation and reporter assays, we found that SRF binds to the miR-199a-5p/214-3p gene cluster promoter after HG stimulation. In vitro, in HPMCs, silencing of miR-199a-5p or miR-214-3p inhibited the HG-induced phenotypic transition and cell migration but enhanced cell adhesion, whereas ectopic expression of mimic oligonucleotides had the opposite effects. Both miR-199a-5p and miR-214-3p targeted claudin-2 and E-cadherin mRNAs. In a PD rat model, treatment with an SRF inhibitor silenced miR-199a-5p and miR-214-3p and alleviated HG-PD fluid-induced damage and fibrosis. Overall, this study reveals a novel SRF-miR-199a/miR-214-E-cadherin/claudin-2 axis that mediates damage and fibrosis in PD.

Idiopathic membranous nephropathy in children in China.

Idiopathic membranous nephropathy (IMN), which was the result from non-SLE, non-hepatitis B or C virus infection, is relatively rare in children. We collected 584 pediatric cases of renal biopsy specimens in children hospitalized in Xijing Hospital from Jan 2008 to Dec 2013. We found that IgA nephropathy (27.9%) and Henoch-Schönlein purpura (26.4%) are the most common etiology of pediatric kidney disease, followed by minimal change disease (10.4%). Surprisingly, the prevalence of IMN is very high (7.5%) in pediatric patients. Moreover, the prevalence of IMN is quickly increasing from 4.4% in 2009 to 9.1% in 2013.

Treatment of severe IgA nephropathy: mycophenolate mofetil/prednisone compared to cyclophosphamide/prednisone.

OBJECTIVE: To compare the efficacy of mycophenolate mofetil (MMF)/prednisone to cyclophosphamide (CYC)/prednisone in the treatment of severe IgA nephropathy. METHODS: Patients (n = 84) with severe IgA nephropathy received either MMF/prednisone (MMF group) or CYC/prednisone (CYC group). The MMF induction dose was 1.5 g/d for 6 months and the maintenance dose was 0.75 - 1.0 g/day for 12 months. The CYC induction dose was 0.8 - 1.0 g/month for 6 months and the maintenance dose was 0.8 - 1.0 g/3 months for 12 months. Laboratory tests, clinical remission rate and side effects were investigated. RESULTS: After 18 months of treatment, the total effective rate in the MMF group was significantly higher than that of the CYC group. Patients' 24-hour urinary protein excretion in the MMF group was lower than the CYC group. Patients' plasma albumin and total protein in the MMF group was higher than the CYC group. MMF and prednisone reduced serum lipids, while in the CYC group serum lipids remained unchanged. There was also a lower incidence of adverse effects in the MMF group (4.76%) than in the CYC group (26.2%). CONCLUSION: Combination therapy with MMF and prednisone for severe IgA nephropathy achieved a higher remission rate compared to treatment with CYC and prednisone. This therapy also reduced the 24-hour urinary protein and serum lipids while increasing plasma albumin and improving renal function. The incidence of adverse effects was significantly lower in the MMF group compared to the CYC group. *These authors have contributed equally to this work.

Acupuncture Alleviates Colorectal Hypersensitivity and Correlates with the Regulatory Mechanism of TrpV1 and p-ERK.

Here we used a mouse model of zymosan-induced colorectal hypersensitivity, a similar model of IBS in our previous work, to evaluate the effectiveness of the different number of times of acupuncture and elucidate its potential mechanism of EA treatment. Colorectal distension (CRD) tests show that intracolonic zymosan injection does, while saline injection does not, induce a typical colorectal hypersensitivity. EA treatment at classical acupoints Zusanli (ST36) and Shangjuxu (ST37) in both hind limbs for 15 min slightly attenuated and significantly blunted the hypersensitive responses after first and fifth acupunctures, respectively, to colorectal distention in zymosan treatment mice, but not in saline treatment mice. Western blot results indicated that ion channel and TrpV1 expression in colorectum as well as ERK1/2 MAPK pathway activation in peripheral and central nerve system might be involved in this process. Hence, we conclude that EA is a potential therapeutic tool in the treatment and alleviation of chronic abdominal pain, and the effectiveness of acupuncture analgesia is accumulative with increased number of times of acupuncture when compared to that of a single time of acupuncture.

Expression of XBP1s in bone marrow stromal cells is critical for myeloma cell growth and osteoclast formation.

BM stromal cells (BMSCs) are key players in the microenvironmental support of multiple myeloma (MM) cell growth and bone destruction. A spliced form of the X-box-binding protein-1 (XBP1s), a major proximal effector of unfolded protein response signaling, is highly expressed in MM cells and plays an indispensable role in MM pathogenesis. In the present study, we found that XBP1s is induced in the BMSCs of the MM microenvironment. XBP1s overexpression in healthy human BMSCs enhanced gene and/or protein expression of VCAM-1, IL-6, and receptor activator of NF-κB ligand (RANKL), enhancing BMSC support of MM cell growth and osteoclast formation in vitro and in vivo. Conversely, deficiency of XBP1 in healthy donor BMSCs displayed a range of effects on BMSCs that were opposite to those cells with overexpression of XBP1s. Knock-down of XBP1 in MM patient BMSCs greatly compromised their increased VCAM-1 protein expression and IL-6 and RANKL secretion in response to TNFα and reversed their enhanced support of MM-cell growth and osteoclast formation. Our results demonstrate that XBP1s is a pathogenic factor underlying BMSC support of MM cell growth and osteoclast formation and therefore represents a therapeutic target for MM bone disease.

Contributions of blood pressure to proteinuria and renal function in the puerperium.

BACKGROUND: Increasing evidence indicates that hypertension in pregnancy plays a key role in cardiovascular disease. However, the correlation between blood pressure (BP) and proteinuria as well as renal function in the puerperium has not been established. PATIENTS: We evaluated the estimated glomerular filtration rate (eGFR) and 24-h urine total protein excretion (TPE) during the second postpartum week in 852 pregnant women with normal BPs and 114 pre-eclamptic women with BPs > or =140/90 mmHg. The 852 pregnant women with normal BPs were divided into two groups based on the mean arterial pressure (MAP): 684 subjects with an MAP<90 mmHg and 168 subjects with an MAP> or =90 mmHg. RESULTS: The eGFR was significantly decreased in pre-eclamptic women (112+/-41 ml/min/1.73 m(2)) compared with healthy women with an MAP<90 mmHg (131+/-35 ml/min/1.73 m(2), p<0.01) and an MAP> or =90 mmHg (128+/-34 ml/min/1.73 m(2), p<0.01), while the TPE was significantly increased compared with healthy women with an MAP<90 mmHg and an MAP> or =90 mmHg (1790+/-1422 vs 124+/-148 and 255+/-427 mg/24 h, respectively; p<0.001). Although the eGFR did not reveal a difference between the two groups of healthy women (131+/-35 vs 128+/-34 ml/min/1.73 m(2), p>0.05), the TPE was significantly higher in subjects with an MAP> or =90 mmHg than in subjects with an MAP<90 mmHg (255+/-427 vs 124+/-148 mg, p=0.004). CONCLUSIONS: Pre-eclampsia induces significant renal injury characterized by an elevation of TPE and a reduction in GFR. BP is closely related to urinary protein excretion, even in healthy women (BP <140/90 mmHg) in the puerperium.

Use of genetic immunization to generate a high-level antibody against rat dicarboxylate transporter.

BACKGROUND: Rat dicarboxylate transporter (SDCT1), expressed in renal tubular epithelial cells, plays a key role in regulating blood and urinary citrate level by reabsorbing citrate from the lumen. Antibodies against this transporter are very important for investigating its expression and function. With the cytokine gene as a molecular adjuvant, genetic immunization-based antibody production offers several advantages compared with current methods. This study aimed, by genetic immunization, to produce a high-specificity antibody against SDCT1. METHODS: We fused a high-antigenicity fragment of SDCT1 to the plasmid pBQAP-TT containing T-cell epitopes and flanking regions from tetanus toxin. Mice were immunized by gene-gun immunization with recombinant plasmid and two other adjuvant plasmids that express granulocyte/macrophage colony-stimulating factor and FMS-like tyrosine kinase 3 ligand, respectively. The titer of the antibody was detected by enzyme-linked immunosorbent assay (ELISA). Specificity of the antibody was identified with SDCT1 native protein in rat kidney by Western blot analysis and immunohistochemistry, and with SDCT1 protein expressed on Xenopus oocytes plasma membranes by immunofluorescence. RESULTS: ELISA measurements showed that the antibody titer was 1:32,000. The native protein of SDCT1 in rat kidney can be recognized by this antibody with Western blot analysis and immunohistochemistry. Immunofluorescence showed that this antibody also recognized SDCT1 protein targeted to Xenopus oocytes plasma membranes into which SDCT1 full-length cRNA was injected. CONCLUSION: Generation of a high-specificity immunoglobulin G antibody against SDCT1 by genetic immunization has provided an important tool for the study of citrate transport.

Aldosterone induces collagen synthesis via activation of extracellular signal-regulated kinase 1 and 2 in renal proximal tubules.

AIM: Aldosterone plays a crucial role in renal fibrosis by inducing mesangial cell proliferation and promoting collagen synthesis in renal fibroblasts. However, renal proximal tubule involvement in aldosterone-induced collagen synthesis has not yet been identified. The aim of this study was to examine the potential role of aldosterone in collagen expression and its possible mineralocorticoid receptor (MR)-dependent pathway, mediated by activation of extracellular signal-regulated kinase 1 and 2 (ERK1/2) in cultured human renal proximal tubular epithelial (HKC) cells. METHODS: After HKC cells were stimulated by aldosterone with different concentrations for various time and periods, the gene expression and protein synthesis of collagen I, II, III and IV were measured by real-time polymerase chain reaction and western blot, respectively. ERK1/2 activation, alpha-smooth muscle actin (alpha-SMA), and E-cadherin were also detected by western blot. RESULTS: Aldosterone can increase ERK1/2 phosphorylation of human renal proximal tubular epithelial cells in a time- and dose-dependent manner. Although aldosterone had no effect on collagen I and II expression, it increased expression of alpha-SMA and collagen III and IV and decreased that of E-cadherin in HKC cells after 48 h. These effects could be prevented by a ERK pathway inhibitor, U0126, or by a selective MR antagonist, spironolactone. CONCLUSION: The results suggest that aldosterone plays a pivotal role in tubulointerstitial fibrosis by promoting tubular epithelial-mesenchymal transition and collagen synthesis in proximal tubular cells. The process is MR-dependent, and mediated by ERK1/2 mitogen-activated protein kinase pathway.

Development of high-specificity antibodies against renal urate transporters using genetic immunization.

Recently three proteins, playing central roles in the bidirectional transport of urate in renal proximal tubules, were identified: two members of the organic anion transporter (OAT) family, OAT1 and OAT3, and a protein that designated renal urate-anion exchanger (URAT1). Antibodies against these transporters are very important for investigating their expressions and functions. With the cytokine gene as a molecular adjuvant, genetic immunization-based antibody production offers several advantages including high specificity and high recognition to the native protein compared with current methods. We fused high antigenicity fragments of the three transporters to the plasmids pBQAP-TT containing T-cell epitopes and flanking regions from tetanus toxin, respectively. Gene gun immunization with these recombinant plasmids and two other adjuvant plasmids, which express granulocyte/ macrophage colony-stimulating factor and FMS-like tyrosine kinase 3 ligand, induced high level immunoglobulin G antibodies, respectively. The native corresponding proteins of URAT1, OAT1 and OAT3, in human kidney can be recognized by their specific antibodies, respectively, with Western blot analysis and immunohistochemistry. Besides, URAT1 expression in Xenopus oocytes can also be recognized by its corresponding antibody with immuno-fluorescence. The successful production of the antibodies has provided an important tool for the study of UA transporters.

NANOG changes in mouse kidneys with age.

NANOG is essential for mouse and human embryonic stem cell (ESC) pluripotency and selfrenewal. It is also expressed in several adult murine tissues as shown by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. However, human NANOG transcripts have been isolated from adult bone marrow (EST; GenBank accession no. BF893620). Here, we study the NANOG gene expression profile in isolated mouse renal papillary cells by Northern blot and RT-PCR. The whole RNA of mouse renal cells was obtained from fresh renal tissues, renal tissues infused by phosphate-buffered saline (PBS), and isolated renal papillary cells of mouse, respectively, as well as the renal papillary tissue from 18.5 days postcoitum (d.p.c.; fetal), 1-2-week-old (young), 1-8-month-old (adult), and 24-month-old (aging) mice. Our analysis shows that a very low expression level was detected in mouse renal tissues, and the renal papillary cells express more than other tissues as determined with Northern blot and RT-PCR. These data suggest that the kidney has its own cells expressing NANOG, and loss of NANOG expression occurs in an age-dependent manner in the kidney, either due to developmental factors or aging, particularly in renal papillary tissue.

Generation of mouse anti-human urate anion exchanger antibody by genetic immunization and its identification.

BACKGROUND: Human urate anion exchanger (hURAT1) as a major urate transporter expressed on renal tubular epithelial cells regulates blood urate level by reabsorbing uric acid. Antibody is an important tool to study hURAT1. This study aimed, by genetic immunization, to produce mouse anti-hURAT1 polyclonal antibody with high throughput and high specificity and to detect the location of hURAT1 in human kidney. METHODS: Human renal total RNA was isolated and the entire cDNA of hURAT1 was amplified by RT-PCR. The sequence of intracellular high antigenicity fragment (A280 to R349) was chosen by prediction software of protein antigenicity, and its cDNA was amplified from cDNA of hURAT1, and then cloned into pBQAP-TT vector to construct recombinant plasmid pBQAP-TT-hURAT1-210 for genetic immunization. Mice were inoculated with this recombinant plasmid and two other adjuvant plasmids, pCMVi-GMCSF and pCMVi-Flt3L, which helped to enhance the antibody's generation. After four weeks, the mice were sacrificed to obtain the anti-hURAT1 antibody from serum. The antibody was identified by western blot analysis and immunohistochemistry. At the same time, rabbit anti-hURAT1 antibody was produced by protein immunization. The specificity and efficiency between the rabbit and mouse anti-hURAT1 antibody were compared by western blot analysis and immunohistochemistry. RESULTS: The entire cDNA of hURAT1 and cDNA of its intracellular high immunogenic fragment were amplified successfully. Recombinant plasmid pBQAP-TT-hURAT1-210 for genetic immunization was confirmed by restriction digestion and sequencing. Both the mouse anti-hURAT1 antibody and rabbit anti-hURAT1 antibody recognized 58 kD hURAT1 and 64 kD glycosylated hURAT1 protein bands in western blot. Immunohistochemically, hURAT1 was located at the brush border membrane of renal proximal tubular cells. In addition, the throughput and specificity of the mouse anti-hURAT1 antibody were higher than those of the rabbit anti-hURAT1 antibody. CONCLUSION: Genetic immunization can generate anti-hURAT1 polyclonal antibody of high throughput and specificity.