The E3 Ligase TRIM25 Impairs Apoptotic Cell Death in Colon Carcinoma Cells via Destabilization of Caspase-7 mRNA: A Possible Role of hnRNPH1.

Therapy resistance is still a major reason for treatment failure in colorectal cancer (CRC). Previously, we identified the E3 ubiquitin ligase TRIM25 as a novel suppressor of caspase-2 translation which contributes to the apoptosis resistance of CRC cells towards chemotherapeutic drugs. Here, we report the executioner caspase-7 as being a further target of TRIM25. The results from the gain- and loss-of-function approaches and the actinomycin D experiments indicate that TRIM25 attenuates caspase-7 expression mainly through a decrease in mRNA stability. The data from the RNA pulldown assays with immunoprecipitated TRIM25 truncations indicate a direct TRIM25 binding to caspase-7 mRNA, which is mediated by the PRY/SPRY domain, which is also known to be highly relevant for protein-protein interactions. By employing TRIM25 immunoprecipitation, we identified the heterogeneous nuclear ribonucleoprotein H1 (hnRNPH1) as a novel TRIM25 binding protein with a functional impact on caspase-7 mRNA stability. Notably, the interaction of both proteins was highly sensitive to RNase A treatment and again depended on the PRY/SPRY domain, thus indicating an indirect interaction of both proteins which is achieved through a common RNA binding. Ubiquitin affinity chromatography showed that both proteins are targets of ubiquitin modification. Functionally, the ectopic expression of caspase-7 in CRC cells caused an increase in poly ADP-ribose polymerase (PARP) cleavage concomitant with a significant increase in apoptosis. Collectively, the negative regulation of caspase-7 by TRIM25, which is possibly executed by hnRNPH1, implies a novel survival mechanism underlying the chemotherapeutic drug resistance of CRC cells. The targeting of TRIM25 could therefore offer a promising strategy for the reduction in therapy resistance in CRC patients.

Sphingosine 1-Phosphate Receptor 5 (S1P5) Knockout Ameliorates Adenine-Induced Nephropathy.

S1P and its receptors have been reported to play important roles in the development of renal fibrosis. Although S1P5 has barely been investigated so far, there are indications that it can influence inflammatory and fibrotic processes. Here, we report the role of S1P5 in renal inflammation and fibrosis. Male S1P5 knockout mice and wild-type mice on a C57BL/6J background were fed with an adenine-rich diet for 7 days or 14 days to induce tubulointerstitial fibrosis. The kidneys of untreated mice served as respective controls. Kidney damage, fibrosis, and inflammation in kidney tissues were analyzed by real-time PCR, Western blot, and histological staining. Renal function was assessed by plasma creatinine ELISA. The S1P5 knockout mice had better renal function and showed less kidney damage, less proinflammatory cytokine release, and less fibrosis after 7 days and 14 days of an adenine-rich diet compared to wild-type mice. S1P5 knockout ameliorates tubular damage and tubulointerstitial fibrosis in a model of adenine-induced nephropathy in mice. Thus, targeting S1P5 might be a promising goal for the pharmacological treatment of kidney diseases.

Consistent alteration of chain length-specific ceramides in human and mouse fibrotic kidneys.

BACKGROUND: Several studies revealed alterations of single sphingolipid species, such as chain length-specific ceramides, in plasma and serum of patients with kidney diseases. Here, we investigated whether such alterations occur in kidney tissue from patients and mice suffering from renal fibrosis, the common endpoint of chronic kidney diseases. METHODS: Human fibrotic kidney samples were collected from nephrectomy specimens with hydronephrosis and/or pyelonephritis. Healthy parts from tumor nephrectomies served as nonfibrotic controls. Mouse fibrotic kidney samples were collected from male C57BL/6J mice treated with an adenine-rich diet for 14 days or were subjected to 7 days of unilateral ureteral obstruction (UUO). Kidneys of untreated mice and contralateral kidneys (UUO) served as respective controls. Sphingolipid levels were detected by LC-MS/MS. Fibrotic markers were analyzed by TaqMan® analysis and immunohistology. RESULTS: Very long-chain ceramides Cer d18:1/24:0 and Cer d18:1/24:1 were significantly downregulated in both fibrotic human kidney cortex and fibrotic murine kidney compared to respective control samples. These effects correlate with upregulation of COL1α1, COL3α1 and αSMA expression in fibrotic human kidney cortex and fibrotic mouse kidney. CONCLUSION: We have shown that very long-chain ceramides Cer d18:1/24:0 and Cer d18:1/24:1 are consistently downregulated in fibrotic kidney samples from human and mouse. Our findings support the use of in vivo murine models as appropriate translational means to understand the involvement of ceramides in human kidney diseases. In addition, our study raises interesting questions about the possible manipulation of ceramide metabolism to prevent progression of fibrosis and the use of ceramides as potential biomarkers of chronic kidney disease.

How to Prevent Renal Cachexia? A Clinical Randomized Pilot Study Testing Oral Supplemental Nutrition in Hemodialysis Patients With and Without Human Immunodeficiency Virus Infection.

OBJECTIVE: End-stage renal disease associates with catabolism and sarcopenia. Hypothetically, peroral supplemental nutrition over 6 months prevents catabolism in hemodialysis patients. DESIGN: Prospective randomized pilot study (ClinicalTrials.gov Identifier: NCT00687050). SUBJECTS: Twenty-three hemodialysis patients (15 males and 7 females) with or without human immunodeficiency virus (HIV) infection of 2 ambulatory hemodialysis centers. INTERVENTION: HIV-positive hemodialysis patients (n = 7, Group 1) were started on supplemental nutrition drinks (250 kcal/day), HIV-negative hemodialysis patients (n = 16, Group 2) were randomized to supplemental nutrition drinks (250 kcal/day) or received none. MAIN OUTCOME MEASURES: Body impedance analysis, anthropometric measures, magnetic resonance imaging results for mid-iliopsoas muscle cross-sectional area and laboratory parameters including albumin, cytokines at baseline, and at 6 months follow-up. RESULTS: Seven patients in Group 1 (mean age: 50.6 ± 9.6 years) and 16 patients in Group 2 (mean age: 54.0 ± 13.3 years) were recruited. Serum creatinine (Group 1: 6.4 ± 3.0 mg/dL; Group 2: 10.7 ± 2.5 mg/dL; P < .01), Body impedance analysis-derived phase angle alpha (Group 1: 5.1 ± 1.2; Group 2: 6.9 ± 1.6; P < .01), mid-arm circumference (Group 1: 26.1 ± 1.3 cm; Group 2: 29.6 ± 2.4 cm; P < .01) were less in Group 1 versus Group 2 patients at baseline suggesting that HIV-positive hemodialysis patients had a poorer nutritional status at baseline. At 6-month follow-up, mortality was higher in Group 1 patients (29%) than in Group 2 patients (6%). There was no significant treatment effect on nutritional status in survivors of Group 1 or in the supplemental nutrition arm of Group 2 when compared with baseline or to untreated controls. CONCLUSIONS: A new oral supplemental nutrition over 6 months had no treatment effect in surviving HIV-positive hemodialysis patients or in maintenance hemodialysis patients without HIV infection. The limitations of this study were small study size and unexpected high mortality among HIV-positive hemodialysis patients.

CTGF Is Expressed During Cystic Remodeling in the PKD/Mhm (cy/+) Rat Model for Autosomal-Dominant Polycystic Kidney Disease (ADPKD).

Connective tissue growth factor (CTGF, also named CCN2) plays an important role in the development of tubulointerstitial fibrosis, which most critically determines the progression to end-stage renal failure in autosomal-dominant polycystic kidney disease (ADPKD), the most common genetically caused renal disease. We determined CTGF expression in a well-characterized animal model of human ADPKD, the PKD/Mhm (cy/+) rat. Kidneys of 12 weeks old (cy/+) as well as (+/+) non-affected rats were analyzed for CTGF RNA and protein expression by RT-PCR, Northern and Western blot analyses, in situ hybridization, and IHC. Besides the established expression of CTGF in glomerular cells in kidneys of wild-type (+/+) animals, in (cy/+) rats, CTGF mRNA and protein were robustly expressed in interstitial, stellate-shaped cells, located in a scattered pattern underlying the cystic epithelium and in focal areas of advanced tubulointerstitial remodeling. Renal CTGF mRNA and protein expression levels were significantly higher in (cy/+) rats compared with their (+/+) littermates. Detection of CTGF expression in cells adjacent to cystic epithelium and in areas of marked fibrosis suggests a role in the local response to cyst development and indicates that CTGF may be a relevant factor contributing to tubulointerstitial fibrosis in polycystic kidney disease.

Kidney Injury Molecule-1 Is Specifically Expressed in Cystically-Transformed Proximal Tubules of the PKD/Mhm (cy/+) Rat Model of Polycystic Kidney Disease.

Expression of kidney injury molecule-1 (Kim-1) is rapidly upregulated following tubular injury, constituting a biomarker for acute kidney damage. We examined the renal localization of Kim-1 expression in PKD/Mhm (polycystic kidney disease, Mannheim) (cy/+) rats (cy: mutated allel, +: wild type allel), an established model for autosomal dominant polycystic kidney disease, with chronic, mainly proximal tubulointerstitial alterations. For immunohistochemistry or Western blot analysis, kidneys of male adult heterozygously-affected (cy/+) and unaffected (+/+) littermates were perfusion-fixed or directly removed. Kim-1 expression was determined using peroxidase- or fluorescence-linked immunohistochemistry (alone or in combination with markers for tubule segments or differentiation). Compared to (+/+), only in (cy/+) kidneys, a chronic expression of Kim-1 could be detected by Western blot analysis, which was histologically confined to an apical cellular localization in areas of cystically-transformed proximal tubules with varying size and morphology, but not in distal tubular segments. Kim-1 was expressed by cystic epithelia exhibiting varying extents of dedifferentiation, as shown by double labeling with aquaporin-1, vimentin or osteopontin, yielding partial cellular coexpression. In this model, in contrast to other known molecules indicating renal injury and/or repair mechanisms, the chronic renal expression of Kim-1 is strictly confined to proximal cysts. Its exact role in interfering with tubulo-interstitial alterations in polycystic kidney disease warrants future investigations.

Pretransplant human leukocyte antigen antibodies detected by single-antigen bead assay are a risk factor for long-term kidney graft loss even in the absence of donor-specific antibodies.

Clinical relevance of ELISA- and single-antigen bead assay (SAB)-detected pretransplant HLA antibodies (SAB-HLA-Ab) for kidney graft survival was evaluated retrospectively in 197 patients transplanted between 2002 and 2009 at the University Clinic Frankfurt. Having adjusted for retransplantation and delayed graft function, a significantly increased risk for death-censored graft loss was found in patients with pretransplant SAB-HLA-Ab [HR: 4.46; 95% confidence interval (CI): 1.47-13.48; P = 0.008]. The risk for increased graft loss was also significant in patients with pretransplant SAB-HLA-Ab but without SAB-detected donor-specific Ab (SAB-DSA) (HR: 4.91; 95% CI of 1.43-16.991; P = 0.012). ELISA was not sufficient to identify pretransplant immunized patients with an increased risk for graft loss. In immunized patients, graft loss was predominantly present in patients who received transplants with a mismatch on the HLA-DR locus. In conclusion, even if our study is limited due to small sample size, the results show an increased risk for long-term graft loss in patients with pretransplant SAB-HLA, even in the absence of DSA. SAB-HLA-Ab-positive patients, being negative in ELISA or CDC assay, might profit from a well-HLA-DR-matched graft and intensified immunosuppression.

Short-term preconditioning enhances the therapeutic potential of adipose-derived stromal/stem cell-conditioned medium in cisplatin-induced acute kidney injury.

The development of new strategies to preserve renal function after acute kidney injury (AKI) is necessary due to limited clinical intervention options. The organ-protective effects of mesenchymal stromal/stem cells (MSCs) and their conditioned medium (CM) have been investigated demonstrating that both separately promoted tubular recovery and ameliorated the outcome of AKI. Nevertheless, strategies to optimise the regenerative potential of both are highly needed. Here we investigated the effects of CM from adipose-derived MSCs (ASCs) preincubated in a hypoxic environment (Hyp). Protective factors were investigated by PCR analysis and a protein array in vitro. The expression of 64 of the 308 proteins assayed was found to be more than two-fold increased after Hyp. CM of Hyp-pretreated ASCs (pCM) was used to enhance regeneration in a mouse model of cisplatin-induced AKI (cisAKI). Renal function was assessed by measurements of markers for AKI and serum cytokine levels. The pCM significantly ameliorated serum creatinine and neutrophil gelatinase-associated lipocalin values, and also the levels of inflammatory cytokines IL-1ß and IL-6 in the serum of mice with AKI. Our work clearly showed that a Hyp preconditioning significantly increases the release of protective factors in ASCs and enhances the therapeutic effects of CM in cisAKI in mice.

Upper and lower urinary tract infections can be detected early but not be discriminated by urinary NGAL in adults.

PURPOSE: We investigated whether the recently established biomarkers of acute kidney injury, neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule-1 (KIM-1), may help to diagnose acute urinary tract infections (UTI) in adults and are able to distinguish between upper or lower localization. METHODS: NGAL levels were measured in blood and urine, and KIM-1 concentrations in urine of 97 subjects. We recruited age- and gender-matched groups of 30 patients with acute upper UTI and 29 patients with acute lower UTI as well as 38 healthy controls. NGAL and KIM-1 were determined by ELISA, serum and urine creatinine applying the Jaffé's method. RESULTS: Urinary NGAL (uNGAL) was significantly increased in patients with upper as well as with lower UTI compared with the healthy controls (P < 0.01; P < 0.05). Accordingly, uNGAL normalized on urinary creatinine (uNGAL/uCrea) was markedly higher in patients with lower UTI compared with the control group (P < 0.05), and uNGAL/uCrea levels were still raised in patients with upper UTI, though not reaching statistical significance (P = 0.07). However, neither uNGAL nor uNGAL/uCrea levels displayed significant differences between patients with upper or lower UTI (P = 0.75; P = 0.97). uKIM-1 levels were close to the detection limit and too low to reliably reveal differences between the three groups. CONCLUSIONS: While uKIM-1 seems not to serve as a helpful biomarker in this setting, increased levels of uNGAL indicate inflammatory processes in the urinary tract in adults. However, the determination of uNGAL levels does not allow to differentiate between upper and lower UTI.

Serum and urinary NGAL but not KIM-1 raises in human postrenal AKI.

BACKGROUND: We examined the value of the novel acute kidney injury (AKI) markers neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule-1 (KIM-1) in acute postrenal impairment. These biomarkers have been evaluated in prerenal and intrarenal AKI so far, but not in human acute postrenal kidney injury. With regard to multimorbid and critically ill patients the discrimination of different AKI origins often remains a challenge. As the trend goes towards a diagnostic panel of AKI markers, we hereby aim to contribute to evaluate further options of discrimination in an observational case-control study. MATERIALS AND METHODS: Blood and urine samples were obtained from 53 patients with acute obstructive nephropathy secondary to ureteral calculi and 52 age-matched healthy controls. Serum NGAL (sNGAL), urinary NGAL (uNGAL) and urinary KIM-1 (uKIM-1) levels were determined using a commercially available ELISA kit, creatinine applying the Jaffé's method. RESULTS: While urinary levels of KIM-1 were not significantly different between patients with obstructive nephropathy and controls, a striking increase in sNGAL (P < 0·001) and uNGAL (P < 0·01) levels was detected in the obstructive nephropathy group. Within the obstructive nephropathy group, sNGAL (P = 0·01) and uNGAL (P = 0·049) but not uKIM-1 correlated positively with the white blood cell count and uNGAL correlated positively (P = 0·002) with the extent of leucocyturia. CONCLUSIONS: High levels of sNGAL and uNGAL observed in stone-induced acute obstructive nephropathy may represent a valuable marker of postrenal AKI. Low uKIM-1 levels may help to discriminate postrenal AKI events using a panel of markers in this setting.

Cyclosporin A and tacrolimus induce renal Erk1/2 pathway via ROS-induced and metalloproteinase-dependent EGF-receptor signaling.

We previously demonstrated that the widely used immunosuppressive drugs cyclosporin A (CsA) and tacrolimus (FK506), independent of immunophilin binding, can activate profibrogenic transforming growth factor ß (TGFß)/Smad signaling cascades in rat renal mesangial cells (MC). Here we report that both peptidyl-prolyl cis/trans isomerase (PPIase) inhibitors activate the extracellular-signaling regulated kinase (ERK) a member of the mitogen activated protein kinase (MAPK) and induce a rapid and transient increase in ERK phosphorylation. The MEK inhibitor U0126, the reactive oxygen species (ROS) scavenger N-acetyl-cysteine (NAC), a cell-permeant superoxide dismutase (SOD) and stigmatellin, an inhibitor of mitochondrial cytochrome bc1 complex strongly attenuated the increase in ERK1/2 phosphorylation triggered by PPIase inhibitors. Moreover, neutralizing antibodies against heparin binding-epidermal growth factor (HB-EGF), and inhibition of the EGF receptor by either small interfering (si)RNA or AG1478, demonstrate that ERK activation by both PPIase inhibitors is mediated via HB-EGF-induced EGF receptor (EGFR) tyrosine kinase activation. The strong inhibitory effects achieved by GM6001 and TAPI-2 furthermore implicate the involvement of a desintegrin and metalloproteinase 17 (ADAM17). Concomitantly, the PPIase inhibitor-induced ADAM17 secretase activity was significantly reduced by SOD and stigmatellin thus suggesting that mitochondrial ROS play a primary role in PPIase inhibitor-induced and ADAM17-mediated HB-EGF shedding. Functionally, both immunosuppressants caused a strong increase in MC proliferation which was similarly impeded when cells were treated in the presence of NAC, TAPI-2 or AG1478, respectively. Our data suggest that CsA and FK506, via ROS-dependent and ADAM17-catalyzed HB-EGF shedding induce the mitogenic ERK1/2 signaling cascade in renal MC.

ADAM10 is expressed in human podocytes and found in urinary vesicles of patients with glomerular kidney diseases.

BACKGROUND: The importance of the Notch signaling in the development of glomerular diseases has been recently described. Therefore we analyzed in podocytes the expression and activity of ADAM10, one important component of the Notch signaling complex. METHODS: By Western blot, immunofluorescence and immunohistochemistry analysis we characterized the expression of ADAM10 in human podocytes, human urine and human renal tissue. RESULTS: We present evidence, that differentiated human podocytes possessed increased amounts of mature ADAM10 and released elevated levels of L1 adhesion molecule, one well known substrate of ADAM10. By using specific siRNA and metalloproteinase inhibitors we demonstrate that ADAM10 is involved in the cleavage of L1 in human podocytes. Injury of podocytes enhanced the ADAM10 mediated cleavage of L1. In addition, we detected ADAM10 in urinary podocytes from patients with kidney diseases and in tissue sections of normal human kidney. Finally, we found elevated levels of ADAM10 in urinary vesicles of patients with glomerular kidney diseases. CONCLUSIONS: The activity of ADAM10 in human podocytes may play an important role in the development of glomerular kidney diseases.

Angiotensin II induces renal plasminogen activator inhibitor-1 and cyclooxygenase-2 expression post-transcriptionally via activation of the mRNA-stabilizing factor human-antigen R.

Angiotensin (Ang) II-induced fibrosis of the kidney is characterized by the enhanced expression of profibrotic and proinflammatory genes, including the serine protease inhibitor plasminogen activator inhibitor-1 (PAI-1) and cyclooxygenase-2 (COX-2). In addition to transcriptional regulation, both genes are subject to post-transcriptional control by AU-rich destabilizing elements that reside within the 3' untranslated region of the mRNA. We demonstrated that the continuous infusion of AngII in rats induced fibrosis concomitant with a significant increase in glomerular PAI-1 and COX-2 expression levels. Using RNA pull-down assays and electromobility shift assays, we demonstrated the increased binding of the ubiquitous RNA-binding protein human-antigen R (HuR) to the mRNAs of both PAI-1 and COX-2 in the cytoplasmic fractions of renal homogenates from AngII-treated rats. Actinomycin D experiments in rat mesangial cells revealed that AngII stabilizes both mRNAs via HuR as proven by small interfering RNA. Mechanistically, AngII promotes an increase in nucleo-cytoplasmic HuR shuttling, which was blocked by the PKC inhibitor rottlerin and the type-I AngII (AT(1)) receptor antagonist valsartan but was unaffected by both AT(2) receptor antagonists PD123319 and CGP42112. Co-immunoprecipitation revealed that AngII treatment caused an increase in nuclear PKC-delta concomitant with binding to nuclear HuR both in vitro and in vivo. The post-transcriptional regulation of PAI-1 and COX-2 by PKC-delta-dependent HuR shuttling may contribute to the pathogenesis of hypertensive nephrosclerosis triggered by AngII.

Multidrug resistance-related protein 2 genotype of the donor affects kidney graft function.

OBJECTIVES: We tested the effect of kidney-specific multidrug resistance-related protein (MRP2, ABCC2) deficiency on renal organic solute disposition as well as on renal protein and gene expression. Furthermore, we investigated whether a particular kidney donor ABCC2 genotype is associated with delayed graft function in patients. METHODS: A new MRP2-deficient rat strain was established. Renal cross-transplantations were performed between congenic MRP2-deficient and wild-type rats. Renal disposition of MRP2 substrates was investigated in native and transplanted rats. Proteomic analyses and transcriptional profiling were performed in rat kidney graft cortices. Ninety-eight human kidney donor-recipient pairs were genotyped for five ABCC2 polymorphisms. The relationship between delayed graft function and ABCC2 genetic variants in donors and recipients was analyzed by backward stepwise logistic regression. RESULTS: In rats, the absence of renal MRP2 reduced renal bilirubin glucuronide excretion at pathologic plasma concentrations, modified renal p-aminohippurate excretion and did not affect renal morphine-6-glucuronide excretion. Renal MRP2 deficiency led to renal cortical protein or mRNA upregulation of glutathione transferase isoenzymes, glutaredoxin 2, and heme oxygenase-1. In patients, a particular donor ABCC2 genotype was associated with an increased incidence of delayed graft function. CONCLUSION: Kidney graft-specific MRP2 deficiency has mild effects on the renal excretion of some organic solutes under experimental conditions and induces a protein and gene expression pattern indicative of activated antioxidant defense mechanisms. This suggests that MRP2 is a determinant of the redox status in tubular epithelial cells and thus of the susceptibility to renal damage under conditions of treatment with multiple drugs and increased oxygen radical formation.

Characterization of CXCL16 and ADAM10 in the normal and transplanted kidney.

The chemokine CXCL16 plays an important role in the recruitment of leukocytes to sites of inflammation influencing the course of experimental glomerulonephritis. Here we show that human kidneys highly express CXCL16 in the distal tubule, connecting tubule and principal cells of the collecting duct with weak expression in the thick ascending limb of Henle. Beside the membrane localization, a soluble form of CXCL16 can be proteolytically released which acts as a chemotactic factor. In human renal tissue the expression pattern of the disintegrin-like metalloproteinase ADAM10 is similar to that of CXCL16, suggesting ADAM10 can potentially cleave CXCL16 in vivo. When we tested this in primary tubular cells we found that blockade of ADAM10 activity inhibited the IFN-gamma induced release of soluble CXCL16. Acute tubular damage in renal allografts was associated with elevated urinary CXCL16 and this correlated with focally increased apical CXCL16 expression in the distal tubules and collecting ducts. Renal allograft biopsies, with a histopathological diagnosis of acute interstitial rejection, showed increased basolateral ADAM10 expression together with high numbers of infiltrating T cells. Our results suggest that CXCL16 and ADAM10 are involved in the recruitment of T cells to the kidney and play an important role in inflammatory kidney diseases.

Synergistic induction of osteopontin by aldosterone and inflammatory cytokines in mesangial cells.

Hypertensive nephrosclerosis is characterized by activation of the renin-angiotensin-aldosterone system in combination with an inflammatory response characterized by an infiltration of T-cells and mononuclear cells, which release proinflammatory cytokines like IL-1beta/TNFalpha. In various models of experimental hypertensive disease the chemokine osteopontin (OPN) enhances further leukocyte infiltration. Therefore, we investigated the induction of OPN expression in renal mesangial cells (MCs) by aldosterone and the inflammatory cytokines IL-1beta/TNFalpha. Incubation with aldosterone resulted in a time- and concentration-dependent increase in OPN mRNA and protein. OPN mRNA expression followed a biphasic time course with an early increase between 4 and 8 h and the second phase starting at 14 h. The early phase was independent of protein synthesis, indicating a direct effect of aldosterone. Aldosterone-mediated induction of OPN was prevented by spironolactone, indicative of a receptor-mediated aldosterone effect. The mineralocorticoid receptor (MR) was identified in MCs by RT-PCR and immunoprecipitation, and shown to interact with a putative aldosterone-response element of the OPN promoter. The proinflammatory cytokines IL-1beta and TNFalpha only marginally affected OPN expression in MCs. However, coincubation of aldosterone and the cytokines synergistically increased OPN mRNA and protein levels. Since the synergistic effect on OPN mRNA was inhibited by diphenyleneiodonium, we assume an involvement of reactive oxygen species (ROS). We conclude that the chemokine OPN is a target gene of aldosterone in renal MCs, which is activated via the MR, and that proinflammatory cytokines enhance aldosterone-dependent OPN expression. In vivo, this may result in further leukocyte infiltration aggravating hypertensive nephrosclerosis.

Aldosterone induces CTGF in mesangial cells by activation of the glucocorticoid receptor.

BACKGROUND: Aldosterone contributes substantially to cardiac and renal injury by acting on target cells not involved in the regulation of salt and water balance. The profibrotic protein connective tissue growth factor (CTGF) has been identified as one of the target proteins of aldosterone. However, the molecular mechanisms of aldosterone-mediated CTGF induction have not been characterized. METHODS: Mesangial cells were treated with aldosterone or dexamethasone. CTGF expression was characterized at the mRNA and protein level. Translocation of the glucocorticoid receptor (GR) was detected by immunocytochemistry and by Western blotting. RESULTS: Aldosterone and dexamethasone induced CTGF at the mRNA and protein level in a time- and concentration-dependent manner. Specific antagonists of the mineralocorticoid receptor, spironolactone, canrenoate or eplerenone, did not inhibit CTGF induction. However, inhibition of the GR by RU486 prevented dexamethasone-as well as aldosterone-induced CTGF expression, indicating the importance of the GR in aldosterone-mediated regulation of CTGF. This notion was confirmed by translocation of the GR to the nucleus upon stimulation with aldosterone. CONCLUSIONS: CTGF is a functional target of aldosterone in mesangial cells, but aldosterone-induced CTGF gene expression is not directly mediated by the mineralocorticoid receptor.

Distinct immunohistochemical expression of osteopontin in the adult rat major salivary glands.

Osteopontin is a multifunctional protein secreted by epithelial cells of various tissues. Its expression in the adult rat major salivary glands has not yet been studied. We examined osteopontin expression by immunohistochemistry using a well characterized monoclonal antibody. Submandibular glands of young adult male rats (70-100 days old) showed specific expression in secretion granules of granular duct cells but also in cells of the striated ducts and excretory duct. In the major sublingual as well as the parotid gland expression was found solely in the duct system. In addition, a few interstitial-like cells exhibiting very strong immunostaining for osteopontin could be found in either organ. Expression could neither be seen in acinar cells nor in cells of the intercalated ducts. Moreover, in submandibular glands of more aged rats (6- to 7-month old) which show well developed granular convoluted tubules, there was almost exclusive expression of osteopontin in granular duct cells as well as in some interstitial-like cells, but barely in the striated/excretory duct system. Western blot analysis of the submandibular gland showed a specific band migrating at approximately 74 kDa, detectable at both age stages. Osteopontin secreted fom granular duct cells may influence the composition of the saliva, e.g. thereby modulating pathways affecting sialolithiasis. Its expression in striated duct cells may also hint to roles such as cell-cell attachment or cell differentiation. The cell-specific expression detected in the rat major salivary glands differs in part from that reported in mice, human and monkey.

Caspofungin is less nephrotoxic than amphotericin B in vitro and predominantly damages distal renal tubular cells.

BACKGROUND: Caspofungin (CAS) has recently been approved for treatment of invasive aspergillosis. In clinical trials, CAS-induced nephrotoxicity was markedly less pronounced compared to amphotericin B (AmB). Nevertheless, in a recent trial, nephrotoxicity in CAS-treated patients was considerably more pronounced than in preceding studies. Therefore, the aim of this study was to assess toxic effects of CAS on human renal proximal and distal tubular epithelial cells (PTC and DTC) in vitro, and to compare them to those of AmB. METHODS: Cells were isolated from human kidney tissue, and exposed to clinically relevant concentrations of CAS and AmB for 24 h. Total DNA content and cell viability were determined by DAPI staining and a modified MTT assay. For testing of cytotoxicity, LDH activity was measured in cell culture supernatants. To assess apoptotic effects, AnnexinV-binding assay and DAPI staining for detection of fragmented DNA were performed. RESULTS: DTC were more vulnerable towards the antifungal agents than PTC. In contrast to AmB, cell-damaging effects of CAS were less severe. DAPI staining revealed slight and dose-dependent antiproliferative effects of CAS at concentrations reflecting relevant plasma levels. At these concentrations, cell viability, determined by MTT assay, was not decreased in PTC and DTC. LDH release was marginally increased in a dose-dependent manner; apoptosis was not detected. Nevertheless, at CAS concentrations reflecting potential tissue concentrations, cell damaging effects were considerably more pronounced. CONCLUSION: Our results suggest that CAS is less nephrotoxic than AmB in vitro. The antiproliferative and cytotoxic effects of CAS predominantly affect DTC, which seem to be more susceptible to CAS-induced damage.

Prediction of acute renal allograft rejection by urinary monokine induced by IFN-gamma (MIG).

Early diagnosis of acute allograft rejection (AR) is still decisive for long-term renal allograft survival. The aim of this study was to define the role of the chemokine monokine induced by IFN-gamma (MIG) (CXCL9) and IFN-gamma-inducible protein 10 (IP-10) (CXCL10) as early markers of AR in renal transplantation (NTX). In a prospective study, 69 de novo renal transplant recipients were monitored and urine samples were collected after NTX for a median of 29 d. In pH-adjusted urine, MIG and IP-10 were determined by modified ELISA. AR was clinically diagnosed in 15 of 69 recipients and confirmed by biopsy in 14 of 15 AR patients (Banff classification). Corresponding to CXCR3-positive infiltrates in renal tissue, urinary MIG was elevated in 14 of 15 AR patients with a median of 2809 pg/ml (quartile 25% and 75% = 870 and 13,000; n = 15), being significantly (P < 0.0001) different from both nonrejecting allograft patients (NO-AR) (median, 25%, and 75%: 96, 1.0, and 161, n = 54) and healthy controls (median, 25%, and 75%: 144, 19, and 208, n = 13). Urinary MIG predicted AR with a sensitivity of 93% and a specificity of 89%. In AR and NO-AR groups, MIG values correlated well with IP-10 (P < 0.001). MIG values indicated both imminent rejection and response to successful antirejection therapy. MIG was not related to intercurrent infections or other causes for impairment of renal function. In a multivariate analysis, MIG correlated best (P < 0.001) with AR from all AR-associated parameters. In conclusion, urinary MIG serves as a very sensitive and specific predictor for AR, mirrors response to antirejection therapy, and thus may contribute to improved long-term renal allograft survival.