Marc Weber

Heme and the Endothelium EFFECTS OF NITRIC OXIDE ON CATALYTIC IRON AND HEME DEGRADATION BY HEME OXYGENASE

We studied the effects of nitric oxide (NO) on the control of excess cellular heme and release of catalytically active iron. Endothelial cells (ECs) exposed to hemin followed by a NO donor have a ferritin content that is 16% that of cells exposed to hemin alone. Hemin-treated ECs experience a 3.5-fold rise in non-heme, catalytic iron 2 h later, but a hemin rechallenge 20 h later results in only a 24% increase. The addition of a NO donor after the first hemin exposure prevents this adaptive response, presumably due to effects on ferritin synthesis. NO donors were found to reduce iron release from hemin, while hemin accumulated in cells. A NO donor, in a dose-dependent fashion, inhibited heme oxygenase activity, measured by bilirubin production. Using low temperature EPR spectroscopy, heme oxygenase inhibition correlated with nitrosylation of free heme in microsomes. Nitrosylation of cellular heme prevented iron release, for while there was heme oxygenase-dependent release of iron in cells incubated with hemin for 24 h, the addition of a NO donor blocked iron release. This indicates that NO readily nitrosylates intracellular free heme and prevents its degradation by heme oxygenase. Nitrosylation of heme was found to reduce sensitization of cells to oxidative injury.

Renal dysfunction in liver transplant recipients: Evaluation of the critical issues

Major progress has been made in the field of liver transplantation since the first procedure was performed nearly 50 years ago. Despite these improvements, renal dysfunction before and after liver transplantation remains a major complicating factor associated with increased health care costs, morbidity, and mortality. Creatinine‐based estimates of renal function are inaccurate in the setting of end‐stage liver disease and often lead to underdiagnosis and late intervention. This issue is critical in that it is important to understand both the etiology and chronicity of renal dysfunction before liver transplantation because the treatment clearly varies, especially with respect to simultaneous liver‐kidney (SLK) transplantation. Because of the scarcity of available grafts, identifying appropriate candidates for SLK transplantation is crucial. Hepatorenal syndrome is common in liver transplant candidates; however, other etiologies of renal dysfunction need to be considered. Renal dysfunction after liver transplantation is common and may have an acute or chronic presentation. Although calcineurin inhibitors (CNIs) have been associated with post–liver transplant nephrotoxicity, their role may be overestimated, and other contributing etiologies should remain in a clinicians differential diagnosis. Alternatives to CNIs have been evaluated; however, a safe immunosuppressive regimen that achieves the preservation of renal function in liver transplant recipients remains to be established. In this review of the literature, renal dysfunction in the setting of liver transplantation is evaluated, and the critical issues that are barriers to improved outcomes are highlighted. Liver Transpl, 2012.

NITRIC OXIDE DONORS MODULATE FERRITIN AND PROTECT ENDOTHELIUM FROM OXIDATIVE INJURY

Ferritin protects endothelial cells from the damaging effects of iron-catalyzed oxidative injury. Regulation of ferritin occurs through the formation of an iron-sulfur cluster within a cytoplasmic protein, the iron regulatory protein (IRP) that controls ferritin mRNA translation. Nitric oxide has been shown to inhibit iron-sulfur proteins and is present at vascular sites of inflammation; therefore, we undertook a study to examine the influence of nitric oxide on changes in endothelial cell ferritin content in response to iron exposure, and the subsequent effects on susceptibility to oxidative injury. Iron-loaded endothelial cells (EC) exposed to nitric oxide donors synthesize markedly less ferritin. Treatment of EC with a nitric oxide donor increases IRP affinity for ferritin mRNA concomitant with a loss of cytoplasmic aconitase activity in iron-laden EC. Iron-treated EC exposed to NO donors were resistant to oxidative injury despite their low ferritin content when examined 1 h after the treatment period. In contrast, 24 h later, these same cells become sensitive to oxidants, whereas iron-treated EC that are ferritin-rich continue to be resistant. In conclusion, NO inhibits the increase of EC ferritin after exposure to iron but provides short-term protection against oxidants; ferritin, in turn, provides durable cytoprotection by inactivating reactive iron.

Opioids Induce Renal Abnormalities in Tumor-Bearing Mice

Background/Aims: The etiology of renal dysfunction in cancer patients is likely to be multifactorial. A large proportion of these patients receive opioid analgesics, but whether opioids contribute to renal dysfunction remains uncertain. In a murine cancer model, we examined the effects of chronic opioid administration on renal function and pathology, and the molecular mechanisms involved. Methods: C3H/HeJ mice implanted with 2472 tumor cells were treated with either morphine or hydromorphone in clinically relevant doses, or PBS (controls). Renal function was assessed by blood and urine chemistry as well as by measuring mean arterial pressure (MAP) and kidney perfusion. Pathological changes in the kidneys were examined by routine histology. Molecular changes were examined by assessing eNOS, iNOS, HO-1 and COX-2 expression in whole-kidney lysates by Western immunoblotting, and cellular colocalization of these enzymes was determined using immunofluorescence microscopy. Results:Three weeks of opioid treatment resulted in increased kidney weight, elevated BUN and proteinuria, and decreased MAP. This was accompanied by histological abnormalities including glomerular enlargement, hypercellularity, peritubular congestion, vasodilatation and tubular casts. The vasoregulatory molecules iNOS, eNOS, HO-1 and COX-2 were upregulated in the kidneys. The NOS inhibitor L-NAME prevented the morphine-induced increase in perfusion and kidney weight. Conclusions: The chronic use of clinically relevant doses of opioidsleads to structural kidney abnormalities, upregulates NOS, COX-2 and HO-1, and results in renal dysfunction in a murine model of cancer.

Recurrent Glomerulonephritis Under Rapid Discontinuation of Steroids

Background. Recurrent glomerulonephritis (GN) remains an important cause of kidney allograft loss and whether rapid discontinuation of steroids (RDS) is associated with a higher risk of recurrence is not known. Methods. We studied recurrence rate, and graft and patient survival in four groups of recipients: 216 recipients with GN transplanted under RDS (group 1), 978 concurrent non-GN recipients transplanted under RDS (group 2), 260 historic comparator group transplanted for GN between 1994 and 1999 with steroid maintenance (group 3), and 950 recipients who were also transplanted between 1994 and 1999 for non-GN and also maintained on steroids (group 4). Regression analysis adjusting for donor and recipient factors, steroid and sirolimus use, and also GN type was used to address factors associated with recurrent disease. Results. The 1-, 5-, and 7-year recurrence rate in the GN group under RDS was 6.7%, 13.7%, and 19.2% and in historic GN recipients maintained on steroids it was 2.4%, 3.8%, and 5.3%, respectively (P<0.0001). RDS was associated with a higher adjusted risk of recurrent disease for all GN types (hazard ratio 4.86; 95% confidence interval 2.34–10.07; P<0.0001). Graft and patient survival were similar in the two GN groups and both were highest among all groups. Notably, death-censored graft survival was not different among the groups. Conclusion. Steroid avoidance may be associated with a higher rate of recurrent GN but no apparent increase in risk of graft loss. This group of recipients needs to be studied more carefully, in larger numbers, and for a longer time period.

Morphine induces mesangial cell proliferation and glomerulopathy via κ-opioid receptors

Morphine sulfate (MS) stimulates mesangial cell (MC) proliferation, a process central to development of glomerular disease. The purpose of this study was to examine whether specific opioid receptors (OR) and signal transducer and activators of transcription 3 (STAT3) signaling are associated with MS-induced MC proliferation. C57Bl/6J and OR-specific knockout (KO) mice were treated for up to 6 wk with PBS, MS (0.7-2.14 mg/kg), naloxone (equimolar to MS), or MS+naloxone (n = 6 per group). Glomerular volume and expression of PCNA, Thy1, and ED1/CD68 were analyzed in kidney sections. Cell proliferation and STAT3 phosphorylation were analyzed by bromodeoxyuridine (BrdU) ELISA and Western blot, respectively, in MCs in vitro. MS treatment led to enlarged kidneys and glomerulopathy and naloxone reversed these effects. MS treatment increased glomerular volume in both mu-OR (MOR) KO and delta-OR (DOR) KO mice, but not in kappa-OR (KOR) KO mice. To ascertain that MS-induced glomerulopathy in vivo was due to MC proliferation, we further examined the OR-specific effects of MS in MCs in vitro. MS-induced MC proliferation in vitro was inhibited by KOR-specific nor-BNI, but not by DOR or MOR-specific antagonists naltrindol or CTOP, respectively. KOR-specific agonist U50488H stimulated proliferation of MCs, but DOR-specific agonist DPDPE and MOR-specific agonist DAMGO did not. MS failed to stimulate proliferation of MCs from KOR KO mice. MS and KOR agonists induced STAT3 phosphorylation, and STAT3 inhibitor blocked KOR agonist-induced MC proliferation. We show that MS stimulates glomerulopathy and MC proliferation via KOR and STAT3 signaling.

Quality of life in elderly kidney transplant recipients

To evaluate quality of life (QOL) in kidney transplant recipients aged 65 and older, identify predictors of impaired physical and mental QOL cross‐sectionally and compare QOL over time with that of younger transplant recipients and general population controls.

Morphine promotes renal pathology in sickle mice

Patients with sickle cell disease (SCD) are often treated with opioids for severe pain. Although opioids are known to have renal-specific effects, their role in nephropathy in SCD remains unknown. Because a subset of patients receives opioids for long periods of time, we examined the influence of chronic morphine treatment on mice with pre-existing renal disease expressing varying amounts of sickle hemoglobin. Morphine treatment for 3–6 weeks resulted in a variety of defects in renal morphology observed using light and electron microscopy. Notably, morphine induced glomerular pathology, resulting in increased glomerular volume, mesangial expansion, mesangial cell proliferation, parietal cell metaplasia, podocyte effacement, and microvillus transformation. Cystic tubulopathy and hemeoxygenase-1 expression and activity were also increased in morphine-treated mice. Naloxone, a non-selective opioid receptor (OR) antagonist, ameliorated these effects. Functionally, the urine albumin to creatinine ratio was increased following acute as well as chronic morphine treatment. These results suggest that clinically relevant doses of morphine induce renal pathology and that OR antagonists may be effective for ameliorating morphine-induced renal disease.

Low birthweight and risk of albuminuria in living kidney donors.

Low birthweight is linked to hypertension, chronic kidney disease and even end‐stage renal disease. We hypothesized that living kidney donors born with lower birthweight may be at increased risk of hypertension, albuminuria, or reduced GFR beyond what is typical following uninephrectomy. Two hundred fifty‐seven living kidney donors who donated at the University of Minnesota between 1967 and 2005 underwent iohexol GFR and urinary albumin excretion measurements. Predictors of iohexol GFR <60 mL/min/1.73 m2, albuminuria, and hypertension were examined using logistic regression. Predictors examined include age at GFR measurement, time since donation, BMI, gender, serum creatinine level (at donation and GFR measurement), systolic and diastolic blood pressure, race, and birthweight. The latter was obtained through self‐report and verified through birth certificates and family members. Older age, higher BMI, and time from donation were associated with reduced GFR. Older age and higher BMI were also associated with hypertension. Birthweight was not associated with GFR <60 mL/min/1.73 m2: OR=0.70, 95% CI (0.28, 1.74), p = 0.45 or hypertension: OR=0.92, 95% CI (0.46, 1.84), p = 0.82 but was associated with albuminuria: OR=0.37, 95% CI (0.15, 0.92), p = 0.03. These data further strengthen the link between low birthweight and potential adverse renal outcomes.

Morphine stimulates platelet-derived growth factor receptor-β signalling in mesangial cells in vitro and transgenic sickle mouse kidney in vivo

BACKGROUND Pain and renal dysfunction occur in sickle cell disease. Morphine used to treat pain also co-activates platelet-derived growth factor receptor-β (PDGFR-β), which can adversely affect renal disease. We examined the influence of morphine in mesangial cells in vitro and in mouse kidneys in vivo. METHODS > Mouse mesangial cells treated with 1 μM morphine in vitro or kidneys of transgenic homozygous or hemizygous sickle or control mice (n=3 for each), treated with morphine (0.75, 1.4, 2.14, 2.8, 3.6, and 4.3 mg kg(-1) day(-1) in two divided doses during the first, second, third, fourth, fifth, and sixth weeks, respectively), were used. Western blotting, bromylated deoxy uridine incorporation-based cell proliferation assay, reverse transcriptase-polymerase chain reaction, immunofluorescent microscopy, and blood/urine chemistry were used to analyse signalling, cell proliferation, opioid receptor (OP) expression, and renal function. RESULTS Morphine stimulated phosphorylation of PDGFR-β and mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) to the same extent as induced by platelet-derived growth factor-BB (PDGF-BB) and promoted a two-fold increase in mesangial cell proliferation. The PDGFR-β inhibitor, AG1296, OP antagonists, and silencing of μ- and κ-OP abrogated morphine-induced MAPK/ERK phosphorylation and proliferation by ~100%. Morphine treatment of transgenic mice resulted in phosphorylation of PDGFR-β, MAPK/ERK, and signal transducer and activator of transcription 3 (Stat3) in the kidneys. Morphine inhibited micturition and blood urea nitrogen (BUN) clearance and increased BUN and urinary protein in sickle mice. CONCLUSION Morphine stimulates mitogenic signalling leading to mesangial cell proliferation and promotes renal dysfunction in sickle mice.