Mai Szu Wu

The Leptospira Outer Membrane Protein LipL32 Induces Tubulointerstitial Nephritis-Mediated Gene Expression in Mouse Proximal Tubule Cells

Tubulointerstitial nephritis is a main renal manifestation caused by pathogenic leptospira that accumulate mostly in the proximal tubules, thereby inducing tubular injury and tubulointerstitial nephritis. To elucidate the role of leptospira outer membrane proteins in tubulointerstitial nephritis, outer membrane proteins from pathogenic Leptospira shermani and nonpathogenic Leptospira patoc extracted by Triton X-114 were administered to cultured mouse proximal tubule cells. A dose-dependent increase of monocyte chemoattractant protein-1 (MCP-1), RANTES, nitrite, and tumor necrosis factor-alpha (TNF-alpha) in the culture supernatant was observed 48 h after incubating Leptospira shermani outer membrane proteins with mouse proximal tubule cells. RT competitive-PCR experiments showed that Leptospira shermani outer membrane proteins (0.2 microg/ml) increased the expression of MCP-1, nitric oxide synthase (iNOS), RANTES, and TNF-alpha mRNA by 3.0-, 9.4-, 2.5-, and 2.5-fold, respectively, when compared with untreated cells. Outer membrane proteins extract from avirulent Leptospira patoc did not induce significant effects. The pathogenic outer membrane proteins extract contain a major component of a 32-kD lipoprotein (LipL32), which is absent in the nonpathogenic leptospira outer membrane. An antibody raised against LipL32 prevented the stimulatory effect of Leptospira shermani outer membrane proteins extract on MCP-1 and iNOS mRNA expression in cultured proximal tubule cells, whereas recombinant LipL32 significantly stimulated the expression of MCP-1 and iNOS mRNAs and augmented nuclear binding of nuclear factor-kappaB (NF-kappaB) and AP-1 transcription factors in proximal tubule cells. An antibody raised against LipL32 also blunted the effects induced by the recombinant LipL32. This study demonstrates that LipL32 is a major component of pathogenic leptospira outer membrane proteins involved in the pathogenesis of tubulointerstitial nephritis.

Leptospirosis: An ignored cause of acute renal failure in Taiwan

Leptospirosis, caused by a spirochete, is the most common zoonosis in domestic or wild animals. Animals excrete infected urine in soil or water and may cause human infections through abrased wound, mucosa, conjunctiva, or by swallowing contaminated water. Clinical presentations of leptospirosis are mostly subclinical. Five to ten percent of leptospirosis are fatal, causing fever, hemorrhage, jaundice, and acute renal failure (Weils syndrome). Leptospirosis has been ignored as a cause of acute renal failure in Taiwan. We report two patients with leptospirosis who presented with high fever, abdominal pain, jaundice, and acute renal failure. Patient 1 died on day 12 of admission of multiple organ failure associated with pancytopenia, hypogammaglobulinemia, and reactive hemophagocytosis. Leptospirosis was recognized after death. Patient 2 was admitted with similar presentations 2 weeks later. Penicillin and doxycycline were given early in the course, and azotemia, jaundice, respiratory failure, and aseptic meningitis gradually improved. Renal biopsy showed interstitial nephritis. Several tubular clearance tests showed proximal tubular defect with severe bicarbonate wasting (FeHCO3- 20.9%) and incomplete type II renal tubular acidosis without affecting the distal nephron. After 80 days of treatment, this patient was discharged with recovery of conscious level and renal function. This is the first leptospirosis patient with detailed tubular functional and morphological studies of the kidney. Diagnosis of leptospirosis was made by microscopic agglutination test (MAT) for antibody to leptospira and by polymerase chain reaction (PCR) for leptospira DNA in blood and urine (interrogans serogroup australis in case 1 and Leptospira borgpetersenii serogroup ballum in case 2). Because active surveillance has resulted in 13 cases diagnosed as leptospirosis islandwide thereafter, underestimation and ignorance of leptospirosis as a cause of acute renal failure may occur in Taiwan. Therefore, an area with a low leptospirosis incidence may actually have a very high incidence. Leptospirosis should be suspected in febrile patients with jaundice and renal failure when pathogens cannot be identified by traditional culture for microorganisms.

Inhibition of the P2X7 Receptor Reduces Cystogenesis in PKD

The P2X7 receptor participates in purinergic signaling, which may promote the progression of ADPKD. We examined the effects of a P2X7 receptor antagonist and a P2X7 receptor agonist on cyst development in a zebrafish model of polycystic kidney disease in which we knocked down pkd2 by morpholinos. We used live wt-1b pronephric-specific GFP-expressing zebrafish embryos to directly observe changes in the pronephros. Exposure of pkd2-morphant zebrafish to a P2X7 receptor antagonist (oxidized ATP [OxATP]) significantly reduced the frequency of the cystic phenotype compared with either exposure to a P2X7 receptor agonist (BzATP) or with no treatment (P < 0.01). Histology confirmed improvement of glomerular cysts in OxATP-treated pkd2 morphants. OxATP also reduced p-ERK activity and cell proliferation in pronephric kidneys in pkd2 morphants. Inhibition of P2X7 with an additional specific antagonist (A-438079), and through morpholino-mediated knockdown of p2rx7, confirmed these effects. In conclusion, blockade of the P2X7 receptor reduces cyst formation via ERK-dependent pathways in a zebrafish model of polycystic kidney disease, suggesting that P2X7 antagonists may have therapeutic potential in ADPKD.

Early Hemoperfusion May Improve Survival of Severely Paraquat-Poisoned Patients

Background Thousands of paraquat (PQ)-poisoned patients continue to die, particularly in developing countries. Although animal studies indicate that hemoperfusion (HP) within 2−4 h after intoxication effectively reduces mortality, the effect of early HP in humans remains unknown. Methods We analyzed the records of all PQ-poisoned patients admitted to 2 hospitals between 2000 and 2009. Patients were grouped according to early or late HP and high-dose (oral cyclophosphamide [CP] and intravenous dexamethasone [DX]) or repeated pulse (intravenous methylprednisolone [MP] and CP, followed by DX and repeated MP and/or CP) PQ therapy. Early HP was defined as HP <4 h, and late HP, as HP ≥4 h after PQ ingestion. We evaluated the associations between HP <4 h, <5 h, <6 h, and <7 h after PQ ingestion and the outcomes. Demographic, clinical, laboratory, and mortality data were analyzed. Results The study included 207 severely PQ-poisoned patients. Forward stepwise multivariate Cox hazard regression analysis showed that early HP <4 h (hazard ratio [HR]u200a=u200a0.38, 95% confidence interval (CI) 0.16–0.86; Pu200a=u200a0.020) or HP <5 h (HRu200a=u200a0.60, 95% CI: 0.39–0.92; Pu200a=u200a0.019) significantly decreased the mortality risk. Further analysis showed that early HP reduced the mortality risk only in patients treated with repeated pulse therapy (nu200a=u200a136), but not high-dose therapy (nu200a=u200a71). Forward stepwise multivariate Cox hazard regression analysis showed that HP <4.0 h (HRu200a=u200a0.19, 95% CI: 0.05–0.79; Pu200a=u200a0.022) or <5.0 h (HRu200a=u200a0.49, 95% CI: 0.24–0.98; Pu200a=u200a0.043) after PQ ingestion significantly decreased the mortality risk in repeated pulse therapy patients, after adjustment for relevant variables. Conclusion The results showed that early HP after PQ exposure might be effective in reducing mortality in severely poisoned patients, particularly in those treated with repeated pulse therapy.

Effects of Cyclosporine, Tacrolimus and Rapamycin on Renal Calcium Transport and Vitamin D Metabolism

Background: Abnormalities in mineral metabolism are common complications of organ transplantation. The role of immunosuppressive agents in alteration of mineral metabolism is not clear. Methods: We conducted an animal study to investigate the effects of cyclosporine A (CsA), tacrolimus, and sirolimus on renal calcium, magnesium and vitamin D metabolism. Results: CsA and tacrolimus induced a 2- to 3-fold and 1.6- to 1.8-fold increase in urinary calcium and magnesium excretion, respectively, while rapamycin had no effects on calcium, but doubled the urinary magnesium excretion. CsA and tacrolimus, but not rapamycin, elevated serum 1,25(OH)2 vitamin D without affecting the parathyroid hormone level. CsA and tacrolimus reduced mRNA abundance in TRPV5 (CsA: 64 ± 3% of control; tacrolimus: 50 ± 3%) calbindin-D28k (CsA: 62 ± 4%; tacrolimus: 43 ± 3%), and vitamin D receptor (CsA: 52 ± 3%; tacrolimus: 58 ± 2%, all p < 0.05). Rapamycin did not affect gene expression in any of studied proteins. The immunofluorescence staining study demonstrated a 50% reduction of TRPV5 and calbindin-D28k by CsA and tacrolimus. Conclusion: The suppression of VDR by calcineurin inhibitors is probably the underlying mechanism of renal calcium wasting. In spite of an increased 1,25(OH)2 vitamin D level, the kidney is not able to reserve calcium, suggesting a role of vitamin D resistance that may be related to bone loss.

Leptospiral Outer Membrane Protein Induces Extracellular Matrix Accumulation through a TGF-β1/Smad-Dependent Pathway

Leptospirosis is an underestimated cause of renal failure in Taiwan and elsewhere. The consequence of leptospira-induced acute tubulointerstitial nephritis is tubulointerstitial fibrosis if left untreated. The aim of the study was to examine the effect of an outer membrane protein (OMP) of Leptospira santarosai serovar Shermani on extracellular matrix (ECM) accumulation in proximal tubular cells, HK-2 cells. The addition of Leptospira santarosai serovar Shermani OMP for 72 h led to an increase of type I and type IV collagens, measured by real-time PCR and Western blot analysis in a dose-response manner. After addition of Leptospira santarosai serovar Shermani OMP, active TGF-beta1 secretion was increased by nearly two-fold. The addition of anti-TGF-beta1-neutralizing antibodies attenuated the Leptospira santarosai serovar Shermani OMP-induced type I and type IV collagen production, implicating TGF-beta1 in this process. Overexpression of the dominant negative Smad3 prevented the Leptospira santarosai serovar Shermani OMP-induced increase of type I or type IV collagen production. In conclusion, this study clearly demonstrated the stimulatory effect of Leptospira santarosai serovar Shermani OMP on ECM production by enhancing ECM synthesis, which was mediated by a TGF-beta1/Smad-dependent pathway.

Patient education: An efficient adjuvant therapy for hyperphosphatemia in hemodialysis patients

Background. Hemodialysis and phosphate (P) binder therapy are the major methods to reduce the phosphate level in dialysis patients. However, dietary P restriction is necessary for adequate control. The successful dietary control is based on patient compliance. Patient education is the best method to assure the knowledge and drive of dietary control, which are the key features for patient compliance. The aim of the study is to investigate quantitatively the effect of patient education on serum P levels in hyperphosphatemic hemodialysis patients. Methods. We conducted a prospective self-control study. Fifty hemodialysis patients with a pre-dialysis serum P level greater than 6.0 mg/dL were studied. Intensified patient education was given. Serum P, calcium (Ca), and intact parathyroid hormone (iPTH) were evaluated before and one month after patient education. Results. Thirty-six (72%) patients had improved pre-dialysis P level (pre-education: 7.50 ± 1.33; post-education: 5.85 ± 1.20 mg/dL, p < 0.001) and Cau2009×u2009P product (pre-education: 68.17 ± 12.70; post-education: 54.70 ± 11.87 mg2/dL2, p < 0.001). The effect lasted for at least three months. There is no significant change on calcium levels. The only predictor of a successful patient education is the iPTH level (improved: 348.8 ± 277.6; non-improved: 668.0 ± 674.1 ng/mL, pu2009=u20090.021). Conclusions. Patient education could be helpful and efficient in hyperphosphatemic control in dialysis patients. The patient education should be given before the serum iPTH level getting high.

Glomerular Transforming Growth Factor-β1 mRNA as a Marker of Glomerulosclerosis – Application in Renal Biopsies

As transforming growth factor-beta1 (TGF-beta1) is implicated in the pathogenesis of glomerulosclerosis, the aim of the study was to demonstrate if levels of glomerular TGF-beta1 mRNA in renal biopsies correlated with glomerulosclerosis. Glomeruli were collected by microdissection from renal biopsies in patients with membranous nephropathy, lupus nephritis, diabetic nephropathy, minimal change disease and IgA nephropathy presented by proteinuria when serum creatinine was <3 mg%. Glomerular mRNAs were reverse transcribed and TGF-beta1, alpha2(IV) collagen, beta-actin cDNA quantitated by competitive polymerase chain reaction (PCR). By semiquantitative electron microscopy, a 3.5-fold increase of glomerular TGF-beta1/beta-actin mRNA ratio in the moderate sclerotic group (n = 23, p < 0.01) and a 1.5-fold increase in the mild sclerotic group (n = 22, p < 0.05) were observed when compared to the minimal sclerotic group (n = 12). A concordant increase of glomerular alpha2(IV) collagen mRNA was found with 2.2- and 1.3-fold in moderate and mild sclerotic groups, respectively. The TGF-beta1/beta-actin mRNA ratios were highest in membranous nephropathy (466.4 +/- 133.4, n = 11), followed by lupus nephritis (394.9 +/- 94.8, n = 12) and diabetic nephropathy (333.2 +/- 97.6, n = 10). Patients with minimal change disease(233.1 +/- 54.1, n = 15)and IgA nephropathy(185.3 +/- 39.6, n = 9) had low levels. The degree of glomerulosclerosis in each group followed the TGF-beta1/beta-actin mRNA ratios indicating that the level is the major determinant ofglomerulosclerosis but not the disease entities. Glomerular TGF-beta1/beta-actin mRNA ratio did not correlate with clinical parameters such as the urinary protein excretion and creatinine clearance. These results suggest that glomerular TGF-beta1/beta-actin mRNA ratio may be used as a marker of glomerulosclerosis in renal biopsy to reflect the local sclerotic process.

Rapamycin in patients with chronic renal allograft dysfunction

Abstract:u2002 Purpose:u2002 Nephrotoxicity of calcineurin inhibitors (CNI) complicates the management of chronic renal allograft dysfunction. Rapamycin is a promising immunosuppressive agent free of nephrotoxicity. The effect of conversion from CNI to rapamycin in recipients with chronic allograft dysfunction is still unclear. We investigated the effect of rapamycin in patients with chronic allograft dysfunction.

Impact of HCV infection on first cadaveric renal transplantation, a single center experience

Abstract:u2002 Background:u2002 Controversy still persists regarding the impact of HCV infection on renal transplant recipients. This study aimed to evaluate the effect of anti‐HCV antibody status on patients and grafts of renal transplants at a single center.