Biomarkers of Tolerance in Kidney Transplantation: Are We Predicting Tolerance or Response to Immunosuppressive Treatment?

We and others have previously described signatures of tolerance in kidney transplantation showing the differential expression of B cell-related genes and the relative expansions of B cell subsets. However, in all of these studies, the index group-namely, the tolerant recipients-were not receiving immunosuppression (IS) treatment, unlike the rest of the comparator groups. We aimed to assess the confounding effect of these regimens and develop a novel IS-independent signature of tolerance. Analyzing gene expression in three independent kidney transplant patient cohorts (232 recipients and 14 tolerant patients), we have established that the expression of the previously reported signature was biased by IS regimens, which also influenced transitional B cells. We have defined and validated a new gene expression signature that is independent of drug effects and also differentiates tolerant patients from healthy controls (cross-validated area under the receiver operating characteristic curve [AUC] = 0.81). In a prospective cohort, we have demonstrated that the new signature remained stable before and after steroid withdrawal. In addition, we report on a validated and highly accurate gene expression signature that can be reliably used to identify patients suitable for IS reduction (approximately 12% of stable patients), irrespective of the IS drugs they are receiving. Only a similar approach will make the conduct of pilot clinical trials for IS minimization safe and hence allow critical improvements in kidney posttransplant management.

Impact of proton pump inhibitor use on magnesium homoeostasis: a cross-sectional study in a tertiary emergency department.

BACKGROUND: To date, the use of proton pump inhibitors (PPIs) has been associated with a low risk of hypomagnesaemia and associated adverse outcomes. We hypothesised that a better risk estimate could be derived from a large cohort of outpatients admitted to a tertiary emergency department (ED). METHODS: A cross-sectional study was performed in 5118 patients who had measurements of serum magnesium taken on admission to a large tertiary care ED between January 2009 and December 2010. Hypomagnesaemia was defined as a serum magnesium concentration < 0.75 mmol/l. Demographical data, serum electrolyte values, data on medication, comorbidities and outcome with regard to length of hospital stay and mortality were analysed. RESULTS: Serum magnesium was normally distributed where upon 1246 patients (24%) were hypomagnesaemic. These patients had a higher prevalence of out-of-hospital PPI use and diuretic use when compared with patients with magnesium levels > 0.75 mmol/l (both p < 0.0001). In multivariable regression analyses adjusted for PPIs, diuretics, renal function and the Charlson comorbidity index score, the association between use of PPIs and risk for hypomagnesaemia remained significant (OR = 2.1; 95% CI: 1.54-2.85). While mortality was not directly related to low magnesium levels (p = 0.67), the length of hospitalisation was prolonged in these patients even after adjustment for underlying comorbid conditions (p < 0.0001). CONCLUSION: Use of PPIs predisposes patients to hypomagnesaemia and such to prolonged hospitalisation irrespective of the underlying morbidity, posing a critical concern.

Determinants of haemodialysis access survival.

BACKGROUND: Vascular access patency is of vital importance for patients requiring haemodialysis. This analysis validates potential risk factors and benefits in patients undergoing vascular access procedures. PATIENTS AND METHODS: Vascular access procedures performed over a two-year period were retrospectively analysed. Clinical data and concomitant medication were retrieved from files as were surgical data following a standardized data capture sheet. Outcome parameters were primary (PP) and secondary patency (SP) as well as freedom from repeated revascularization. Minimal follow-up with functioning access was 679 days. RESULTS: During the observation period, 244 patients (mean age 62.2 +/- 0.9 years, 60.7 % male patients, 36.1 % pre-emptive, 31.1 % late referral) underwent vascular accesses procedures. PP and SP were 35.6 % and 45.6 %, respectively, at 540 days. Presence of diabetes mellitus was associated with decreased PP (OR: 0.6, 95 %-CI: 0.3 - 1.0) and SP (OR: 0.4, 95 %-CI: 0.2 - 0.7), whereas female gender was associated with lower SP (OR: 0.6, 95 %-CI: 0.3 - 0.9) and freedom from repeated revascularization rates (OR: 0.6, 95 %-CI: 0.3 - 1.0). In contrast, presence of hyperparathyreoidism was associated with higher SP (OR: 1.7, 95 %-CI: 1.0 - 3.0) and freedom from repeated revascularization (OR: 1.7, 95 %-CI: 1.0 - 3.0) rates. CONCLUSIONS: Haemodialysis access performs worst in patients with diabetes mellitus and in women. The benefit of hyperparathyroidism should be interpreted as hypothesis generating.

Apparent systemic 11ß-dehydroxysteroid dehydrogenase 2 activity is increased in preeclampsia but not in intrauterine growth restriction.

OBJECTIVE: The enzyme 11ß-dehydroxysteroid dehydrogenase 2 (11ß-HSD2) converts active cortisol (F) to inactive cortisone (E). A reduced 11ß-HSD2 activity in the placenta has been demonstrated for prematurity, low birth weight, and preeclampsia. We hypothesized that disturbed placental function rather than a maternal response contributes to decreased 11ßHSD2 activity as reflected by a diminished conversion of F to E. Hence, the aim of the present study was to estimate the systemic activity of 11ß-HSD2 throughout gestation and in pregnancies complicated by preeclampsia (PE) and intrauterine growth restriction (IUGR) by calculating maternal serum F/E ratios. METHODS: A total of 188 maternal serum samples were analyzed for nine glucocorticoid metabolites by gas chromatography-mass spectrometry (GC-MS) and F/E ratios were calculated. Study Group A: In a longitudinal set 33 healthy pregnant women were analyzed at three different time points throughout gestation and one postpartum. Study Group B: Cross-sectionally additional 56 patients were enrolled. We compared patients with PE (N = 14) and IUGR (N = 14) with gestational age matched healthy controls (CTRL = 28). RESULTS: Group A: The apparent 11ß-HSD2 activity dropped in the second trimester being restored to first trimester levels (P value = 0.016). Group B: The 11ß-HSD2 activity was high in PE (P value < 0.05) but not in the IUGR group as compared to CTRL. CONCLUSION: The increased apparent serum 11ß-HSD2 activity observed with advancing gestation in normal pregnancy may reflect an elevated general increase in enzyme activity due to a higher placental mass. The high systemic 11ß-HSD2 activity in PE but not in IUGR however suggests an increased F deactivation in maternal tissue in PE rather than in the placenta since placental insufficiency in the absence of PE does not significantly alter F/E ratio.

[Urinary hormone analysis]. / Hormonanalysen im Urin.

Urinary hormone analysis is applied to detect an altered steroid hormone metabolism, an elevated production of biogenic amines and to non-invasively determine the protein hormone human beta-choriogonadotropin indicating a pregnancy. Occasionally, these determinations need to be complemented by plasma- or serum hormone analysis. Clinical data including current drug therapy and urinary creatinine as reference are required to interpret any urine analysis. Diseases to be investigated by steroid hormone analysis are excess production of a typical or atypical mineralocorticoid active steroid hormones, the hormonal activity of adrenal or ovarian tumors, acne of unknown origin, hirsutism, a PCO-, an adrenogenital or a suspected Cushing syndrome. Biogenic amines should be determined in suspected secondary or refractory arterial hypertension, in case of pheochromocytoma- or paraganglioma-associated symptoms or if a serotonin-producing tumor is suspected. In children genetically determined diseases are the primary background to perform an analysis.

Maternal Serum Lipid, Estradiol, and Progesterone Levels in Pregnancy, and the Impact of Placental and Hepatic Pathologies.

OBJECTIVE: Lipids and steroid hormones are closely linked. While cholesterol is the substrate for (placental) steroid hormone synthesis, steroid hormones regulate hepatic lipid production. The aim of this study was to quantify circulating steroid hormones and lipid metabolites, and to characterize their interactions in normal and pathological pregnancies with a focus on hepatic and placental pathologies. METHODS: A total of 216 serum samples were analyzed. Group A consisted of 32 patients with uncomplicated pregnancies who were analyzed at three different time-points in pregnancy (from the first through the third trimester) and once post partum. Group B consisted of 36 patients (24th to 42nd week of gestation) with pregnancy pathologies (IUGR n = 10, preeclampsia n = 13, HELLP n = 6, intrahepatic cholestasis n = 7) and 31 controls with uncomplicated pregnancies. Steroid profiles including estradiol, progesterone, and dehydroepiandrosterone were measured by GC-MS and compared with lipid concentrations. RESULTS: In Group A, cholesterol and triglycerides correlated positively with estradiol (cholesterol ρ = 0.50, triglycerides ρ = 0.57) and progesterone (ρ = 0.49, ρ = 0.53) and negatively with dehydroepiandrosterone (ρ = - 0.47, ρ = - 0.38). Smoking during pregnancy affected estradiol concentrations, leading to lower levels in the third trimester compared to non-smoking patients (p < 0.05). In Group B, cholesterol levels were found to be lower in IUGR pregnancies and in patients with HELLP syndrome compared to controls (p < 0.05). Steroid hormone concentrations of estradiol (p < 0.05) and progesterone (p < 0.01) were lower in pregnancies with IUGR. DISCUSSION: Lipid and steroid levels were affected most in IUGR pregnancies, while only minor changes in concentrations were observed for other pregnancy-related disorders. Each of the analyzed entities displayed specific changes. However, since the changes were most obvious in pregnancies complicated by IUGR and only minor changes were observed in pregnancies where patients had impaired liver function, our data suggests that placental rather than maternal hepatic function strongly determines lipid and steroid levels in pregnancy.

Sirolimus-associated eyelid edema in kidney transplant recipients.

BACKGROUND: The immunosuppressant sirolimus is effective in preventing acute rejection episodes. So far, unusual edema formation has not been reported as a side effect. METHODS: Two groups of patients with renal transplants, consisting of 11 patients each, were followed for up to 29 months. The immunosuppressive regimen was either sirolimus and prednisone with or without cyclosporine or azathioprine/mycophenolate and prednisone with cyclosporine. Routine follow-up included a thorough clinical investigation. Edema formation was documented photographically. RESULTS: In 5 of the 11 patients treated with sirolimus uni- or bilateral, non-itching, eyelid edema was observed. After discontinuation of sirolimus, lid edema disappeared. The duration until recovery varied from weeks to months. No cause of edema formation other than the treatment with sirolimus was detected. CONCLUSIONS: Severe eyelid edema formation seems to be associated with sirolimus treatment. The underlying mechanism is unknown.

Nitric oxide synthase activity and Doppler parameters in the fetoplacental and uteroplacental circulation in preeclampsia.

OBJECTIVE: We investigated the hypothesis that changes in blood flow in the uteroplacental and fetoplacental circulation in preeclampsia are associated with an abnormality of placental or uterine placental bed nitric oxide (NO) synthesis. METHODS: We measured pulsatility indices on Doppler waveform analysis from uterine and umbilical arteries in 20 patients with preeclampsia and 14 healthy pregnant controls before elective cesarean section. During cesarean section, biopsies from the uterine placental bed and the placenta were taken and the nitric oxide synthase (NOS) activity was measured by the [3H] L-arginine-[3H] L-citrulline conversion assay in these samples. RESULTS: The NOS activity was significantly lower in the uterine placental bed in comparison to the placental tissue (p < 0.01). Placental NOS activity was similar between patients with preeclampsia and healthy controls and in the groups with either a pathological or a normal Doppler flow in the umbilical artery. In the uterine placental bed however, NOS activity from patients with preeclampsia was significantly lower (p < 0.01), whereas the blood flow resistance in the uterine arteries was elevated (p < 0.01) in comparison to healthy controls. CONCLUSIONS: Our data show that pathological Doppler waveforms in the uterine arteries of patients with preeclampsia are paralleled by diminished NOS activity in the uterine placental bed. Therefore, the compromised NO production in the uterine placental bed may play an important role in the impaired uteroplacental blood flow and potentially in some pathological features of preeclampsia such as intervillous thrombosis formation and fetal growth retardation.

[Microhematuria--what next?]. / Mikrohämaturie--Wie weiter?

Asymptomatic microhematuria is a common reason for a urological consultation. Uncertainty prevails as to how meticulous the work-up must be, to not miss relevant or even life-threatening underlying diseases. To date, the Urological Associations have not released any guidelines to which extent patients need to be examined for asymptomatic microhematuria, which therefore is managed individually by each urologist. There are various potential examinations that can be applied, ranging from a clinical examination to a kidney biopsy. After reviewing the literature, an algorithm has been developed, which should assure diagnosis of serious disease and at the same time avoid costly, unpleasant and unnecessary examinations.

[Edema in pregnancy--trivial?]. / Odeme in der Schwangerschaft--banal?

In pregnancy total body water increases. At least 25% of the fluid will be distributed to the interstitial space, ultimately clinically imposing as lower leg edema of pregnant women. Next to a cumulative sodium retention, altered local Starling forces and changes in the hydration of extracellular matrix add to the fluid shift. Edema have to be expected in most of the pregnant women and should not be used to diagnose preeclampsia. Atypical edema localization and local, unilateral edema should cast suspicion of other dangerous complications of pregnancy. Diuretics should be restricted to pulmonary edema of preeclampsia, but these drugs are not to be used to manage edema of pregnancy.

[Rhabdomyolysis]. / Rhabdomyolysen.

Rhabdomyolysis is a severe clinical symptom of variable etiology. Acquired factors of exogenous origin such as traumata and endogenous metabolic disturbances have to be separated from hereditary disease as causative mechanism. Most frequently, exertional stress during hyperthermia, traumatic damage or ethanol abuse are observed. Almost independent of the diverse initial events, the pathogenesis follows a common final pathway with intracellular calcium accumulation and ATP depletion. Clinical symptoms vary. Seldom, the classical triad of muscle pain, weakness, and dark urine is observed. Recurrent episodes should raise suspicion of an inherited disorder. Severe complications are hypovolemia, electrolyte disorders with hyperkalemia and hypocalcemia resulting in life threatening arrhythmias, a compartment syndrome, disseminated intravascular coagulation and acute renal failure, which is frequently oligo-anuric. In combination with often severe underlying disease, renal failure causes death in 1/5 of the patients. The diagnosis is made with the determination of serum creatine kinase and the myoglobin levels in plasma and urine. Therapeutic options are to correct the hypovolemia with sufficient fluid supply, the prevention of oliguria using loop diuretics, alkalinization of the urine, normalization of serum electrolytes with reduction of hyperkaemia, and decompression of compartment syndromes. An underlying disease should be evaluated to initiate specific therapeutical and preventative steps. Avoiding pre-disposing factors by identifying the mechanisms of disease will reduce the occurrence of rhabdomyolysis with its still high mortality.

[Anuria after delivery]. / Anurie nach der Geburt.

During the last decade certain peripartal complications decreased dramatically in industrialized countries. Here, a case of a 27 years old Caucasian primagravida will be presented. In the presence of symptoms of a severe preeclamptic condition with signs of an impending HELP syndrome the patient underwent Caesarean section. Within hours following surgery she developed complete anuria. Nuclear magnetic resonance imaging and histological evaluation of a renal biopsy led to the diagnosis of an acute, bilateral renal cortical necrosis. Besides the preeclamptic condition no further underlying disease was present, in particular no hemolytic-uremic syndrome. Following progress made in modern perinatal management renal cortical necrosis almost disappeared. Yet, in the presence of this disease a significant maternal morbidity and mortality still remains.

PP010. Does aldosterone participate in placental angiogenesis via PLGF?

INTRODUCTION: Angiogenic signals are a vital signal of placental integrity. Aldosterone has recently been shown to enhance placental growth factor (PlGF) expression in the peripheral vasculature [1] and to promote trophoblast growth [2]. The plgf gene possesses a functional mineralocorticoid receptor responsive element in the promoter region. OBJECTIVES: Thus, we hypothesized that aldosterone adapts placental angiogenesis to trophoblast growth by secreting PlGF. METHODS: The human choriocarcinoma cell line BeWo and first and third trimester human primary trophoblasts cells were subjected to several syncytialization signals. Upon visual confirmation, the cultured cells were subjected to either control conditions, the known stimulator forskolin, and increasing amounts of aldosterone (10(-9) to 10(-6)M) with and without the competitive aldosterone receptor blocker spironolactone. After 6 and 24h of incubation, RNA and protein were extracted. PlGF transcripts were quantified by Taqman PCR normalized to several housekeeping genes. Protein expression was quantified by ELISA. RESULTS: PlGF mRNA expression increased 3-fold with forskolin in BeWo cells. In this cell line, aldosterone could slightly stimulate PlGF production. In non-syncytialized primary human first trimester trophoblasts, aldosterone did not exert a specific effect. In contrast, the term primary human trophoblasts did respond with a 2.5-fold increase after incubation with aldosterone (10(-7)M) in the presence of forskolin to allow forming a syncytial layer. PlGF protein was already slightly upregulated following 6h of incubation with aldosterone. CONCLUSION: We concluded that aldosterone does regulate PlGF expression in specified conditions during pregnancy. Inappropriately low aldosterone levels such as in preeclampsia might such not only compromise plasma volume and trophoblast growth but also placental vascularization and systemic PlGF availability. These observations merit further investigation.

OS058. Aldosterone deficiency adversely affects pregnancy outcome in mice.

INTRODUCTION: In pregnancy, plasma volume is expanded due to high aldosterone levels to support placental perfusion and fetal nutrition. Inadequately low aldosterone levels as present in preeclampsia, a life-threatening disease for both mother and child, are discussed to be involved in its pathogenesis or severity. OBJECTIVES: We used aldosterone synthase deficient (AS(-/-)) mice to test whether the absence of aldosterone is sufficient to impair pregnancy or even to cause preeclampsia. METHODS: AS(-/-) and AS(+/+) females were mated with AS(+/+) and AS(-/-) males, respectively, always generating AS(+/-) offspring. Blood pressure was measured by tail cuff, fetal and placental number and size as well as placental histology were assessed. Placental expression of HIF-1αand angiogenic factors was assessed by semiquantitative RT-PCR. RESULTS: With maternal aldosterone deficiency in AS(-/-) mice, systolic blood pressure was low before and further reduced during pregnancy and with no increase in proteinuria. Yet, AS(-/-) had smaller litters due to loss of fetuses as indicated by a high number of necrotic placentas with massive lymphocyte infiltrations at gestational day 18. Surviving fetuses and their placentas from AS(-/-) females were smaller. High salt diet before and during pregnancy increased systolic blood pressure only before pregnancy in both genotypes and reduced blood pressure during late pregnancy as compared to normal salt controls. Litter size from AS(-/-) was slightly improved and the differences in placental and fetal weights between AS(+/+) and AS(-/-) mothers disappeared. Overall an increased placental efficiency was observed in both groups. CONCLUSION: Our results demonstrate that aldosterone deficiency has profound adverse effects on placental function. High dietary salt intake improved placental function and lowered blood pressure in wild-type mice. In this animal model, aldosterone deficiency did not cause preeclampsia.

OS067. VEGF, a novel stimulator of aldosterone production.

INTRODUCTION: Adrenal aldosterone production depends upon capillary integrity. Inadequately explained by increased renin secretion, aldosterone is high in pregnancy, a proangiogenic state. In preeclampsia, low aldosterone levels coincide with disturbed endothelial integrity due to disrupted VEGF signaling. OBJECTIVES: We hypothesized that the stimulation of adrenal aldosterone production is VEGF-sensitive. METHODS: We cultured endothelial cells (EC) in the presence and absence of VEGF. The supernatent was transferred to cultured adrenal cells, either the cell line H295R or isolated primary human adrenal cells from zona glomerulosa. aldosterone synthase mRNA and protein expression, aldosterone synthesis was assessed by adding radioactive labeled precursors or measuring aldosterone in the supernatent by Elisa. Cells were cultured either with angiotensin II (Ang II), VEGF or a combination hereof. Adenovirus-based overexpression of the soluble VEGF receptor type 1 (sFlt-1) was used to simulated conditions of preeclampsia in rats and its effect on the adrenocortical vasculature and circulating aldosterone levels. RESULTS: EC conditioning in the presence of VEGF enhanced aldosterone synthase activity in human adrenocortical cells. VEGF either alone or combined with Ang II increased aldosterone synthase transcription, enzyme availability and aldosterone production in adrenal cells. Neuropilin-1 and VEGF receptor expression differed only for Flt-1 which was present in ECs but not in adrenocortical cells. In contrast to Ang II, VEGF did not upregulate the steroidogenic acute regulatory protein. In line with this observation, Ang II stimulated both aldosterone and cortisol synthesis from progesterone whereas VEGF preferably the former. In rats, overexpression of sFlt-1 which traps VEGF led to adrenocortical capillary rarefaction. Serum aldosterone concentrations inversely correlated with sFlt-1 levels. CONCLUSION: In conclusion, VEGF stimulates aldosterone production indirectly via ECs and directlyin adrenocortical cells a finding explaining the increased aldosterone/renin ratio in normal pregnancy. It is reasonable to assume that the inappropriately low aldosterone availability in preeclampsia is a consequence of the known disturbed VEGF signaling.

Endothelin-1 inhibits cytokine-stimulated transcription of inducible nitric oxide synthase in glomerular mesangial cells.

Endothelin-1 (ET-1) is a potent vasoconstrictor while nitric oxide (NO) has strong vasodilatory effects. Recent studies have indicated that vasoconstrictors and NO may mutually modulate their production and/or activity, thus regulating each other in the context of microcirculatory maintenance. We examined the question whether ET-1 may affect NO formation by controlling the expression of the inducible isoform of the NO synthase (iNOS) in cultured rat glomerular mesangial cells (MCs), as induced by the inflammatory cytokines, tumor necrosis factor-alpha (TNF-alpha) plus interleukin-1 beta (IL-1 beta). We found that ET-1 in MCs markedly reduced cytokine-induced NO production (measured as stable NO2-) and inhibited the expression of iNOS mRNA (Northern blot analysis) and of iNOS protein (Western blotting). Inhibition of cytokine-stimulated iNOS mRNA expression by ET-1 was almost complete at the level of gene transcription while post-transcriptional effects were not detected. The ETA receptor antagonist BQ-123 blocked the inhibitory effect of ET1. The ETA agonist sarafotoxin 6b (S6b) inhibited, while the ETB agonist-sarafotoxin 6c (S6c) did not inhibit cytokine-initiated iNOS transcription in MCs. The results demonstrate that ET-1 can strongly inhibit cytokine induction of iNOS and formation of NO in cultured MCs, and that this action is mediated via the ETA receptor. While the precise mechanism(s) and biological relevance of this ET-1 effect are presently unclear, it is conceivable that down-regulation of iNOS by the vasopressor ET-1 may serve in vivo to prevent massive NO build-up and subsequent vasomotor collapse in the glomerular capillary tuft. This could help to maintain glomerular ultrafiltration in states of endotoxin excess as well as during glomerular formation and action of TNF-alpha and IL-1 beta causing iNOS induction and subsequent overproduction of NO.

3D Ultrasound imaging--a useful non-invasive tool to detect AV fistulas in transplanted kidneys.

BACKGROUND: A precise, non-invasive, non-toxic, repeatable, convenient and inexpensive follow-up of renal transplants, especially following biopsies, is in the interest of nephrologists. Formerly, the rate of biopsies leading to AV fistulas had been underestimated. Imaging procedures suited to a detailed judgement of these vascular malformations are to be assessed. METHODS: Three-dimensional (3D) reconstruction techniques of ultrasound flow-directed and non-flow-directed energy mode pictures were compared with a standard procedure, gadolinium-enhanced nuclear magnetic resonance imaging angiography (MRA) using the phase contrast technique. RESULTS: Using B-mode and conventional duplex information, AV fistulas were localized in the upper pole of the kidney transplant of the index patient. The 3D reconstruction provided information about the exact localization and orientation of the fistula in relation to other vascular structures, and the flow along the fistula. The MRA provided localization and orientation information, but less functional information. Flow-directed and non-flow-directed energy mode pictures could be reconstructed to provide 3D information about vascular malformations in transplanted kidneys. CONCLUSION: In transplanted kidneys, 3D-ultrasound angiography may be equally as effective as MRA in localizing and identifying AV malformations. Advantages of the ultrasound method are that it is cheaper, non-toxic, non-invasive, more widely availability and that it even provides more functional information. Future prospective studies will be necessary to evaluate the two techniques further.

In situ hybridization localization of mRNA encoding inducible nitric oxide synthase in rat kidney.

We used in situ hybridization with a digoxigenin-labeled cRNA for inducible nitric oxide synthase (iNOS) to characterize the intrarenal distribution of iNOS transcripts in normal and lipopolysaccharide (LPS)-treated rats. In normal rats, the S3 segment of the proximal tubule, the cortical and medullary thick ascending limb, the distal convoluted tubule, and the cortical and inner medullary collecting duct were intensely labeled, whereas the thin limbs of Henle, proximal convoluted tubule, outer medullary collecting duct, and medullary interstitial cells were weakly labeled. LPS-treated rats exhibited a similar labeling pattern, but with increased staining of mesangial cells, medullary interstitial cells, and papillary surface epithelium. The renal vasculature, including the afferent arteriole, was not labeled in either group. No cellular labeling was observed when the sections were hybridized with the sense iNOS probe. These results indicate that iNOS mRNA is tonically and differentially expressed along the normal rat nephron and that LPS induces iNOS gene expression in normally quiescent mesangial cells, medullary interstitial cells, and papillary surface epithelium.

Cytokines activate inducible nitric oxide synthase gene transcription in inner medullary collecting duct cells.

The effects of lipopolysaccharide (LPS) and/or inflammatory cytokines on the expression of inducible nitric oxide synthase (iNOS) were studied in mIMCD-3 cells, derived from the murine inner medullary collecting duct. Under basal conditions, the production of nitrite, a stable metabolite of NO, was negligible; however, incubation with tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IF-gamma) for 24 h resulted in a 12-fold increase in nitrite synthesis and the appearance of abundant iNOS mRNA and protein. The induction of nitrite production and iNOS mRNA was time dependent, requiring approximately 8 h for expression of significant levels of nitrite or iNOS mRNA. Coincubation with the transcription inhibitor actinomycin D or the translation inhibitor cycloheximide prevented the cytokine induction of iNOS mRNA and NO production, indicating that synthesis of intermediary proteins stimulated transcription of the iNOS gene. Nuclear run-on transcription demonstrated that the iNOS gene was transcriptionally inactive under basal conditions, but was markedly induced by TNF-alpha and IF-gamma. These results indicate that inflammatory cytokines stimulate NO production in mIMCD-3 cells by activating iNOS gene transcription in a process that requires new protein synthesis.

Role of NF-kappa B in the regulation of inducible nitric oxide synthase in an MTAL cell line.

The effects of cytokines, lipopolysaccharide (LPS), 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP), and pyrrolidine dithiocarbamate (PDTC), an inhibitor of nuclear factor kappa B (NF-kappa B) activation, on inducible nitric oxide synthase (iNOS) expression were studied in the medullary thick ascending limb of Henle's loop cell line ST-1. LPS + interferon-gamma (IF-gamma) promoted a time-dependent increase in nitrite (a NO metabolite) and iNOS mRNA and the appearance of NF-kappa B p50 and p65 in nuclear protein extracts. Actinomycin D but not cycloheximide prevented the LPS + IF-gamma induction of iNOS mRNA and NO synthesis, indicating that iNOS transcriptional activation by LPS + IF-gamma does not require newly synthesized proteins. PDTC inhibited the LPS + IF-gamma induction of NO, iNOS mRNA, and the appearance of NF-kappa B in nuclear protein extracts, suggesting that NF-kappa B mobilization and trans-activation of the iNOS gene mediates this induction. In contrast to other cell types, cycloheximide did not alter iNOS mRNA stability, and 8-BrcAMP did not alter basal or LPS+IF-gamma induced NO production in ST-1 cells.