Analysis of Risk Factors for Allograft Outcome Comparing 2 Kidneys From the Same Donor in Separate Recipients.

BACKGROUND: An analysis of 2 kidney transplants from the same donor at the same center enables us to analyze the influence of risk factors on the outcome of the grafts in different recipients. METHODS: We retrospectively analyzed 88 kidneys from 44 donors that were implanted in 88 recipients at our institution between 2007-2016. We defined unsatisfactory outcome as glomerular filtration rate <30 mL/min/1.73 m2 allograft loss or recipient death within the first year after transplantation. Fifty-three kidneys were allocated and age-matched to donors above the age of 65 years (via Eurotransplant Senior Program or center offer). We compared kidney pairs with satisfactory outcome in both recipients (group A) to pairs with divergent outcome (group B) and unsatisfactory outcome in both recipients (group C). RESULTS: Thirty-four grafts (17 donors) had a satisfactory outcome for both recipients (group A), and 16 grafts (8 donors) had an unsatisfactory outcome for both recipients (group C). Donor age was significantly higher in group C vs group A (67.5 ± 6.7 vs 56.4 ± 16.0 years, P = .010). The 19 donors donating 1 kidney with satisfactory and the other with unsatisfactory outcome were 67.4 ± 10.7 years old (group B). A severe surgical complication occurred more often in recipients with an unsatisfactory outcome in comparison to patients with a satisfactory outcome. CONCLUSION: Donor age is an important risk factor for an unsatisfactory outcome, either in one or both kidneys of the same donor.

Outcome and natural course of renal dysfunction in liver transplant recipients with severely impaired kidney function prior to transplantation.

BACKGROUND: Since introduction of the MELD score in the liver allograft allocation system, renal insufficiency has emerged as an increasing problem. Here we evaluated the course of kidney function in patients with advanced renal insufficiency prior to liver transplantation (LT). METHODS: A total of 254 patients undergoing LT at the University Medical Centre Hamburg-Eppendorf (2011-2015) were screened for renal impairment (GFR < 30 ml/min) prior to LT in this observational study. RESULTS: Eighty (32%) patients (median 60 years; M/F: 48/32) had significant renal impairment prior to LT. Median follow-up post-LT was 619 days. Patient survival at 90 days, one year and two years was 76%, 66% and 64%, respectively. Need for dialysis postoperatively but not preoperatively was associated with increased mortality (p < 0.05). Renal function improved in 75% of survivors, but 78% of patients had chronic kidney disease ≥ stage 3 at end of follow-up. Of eight (16%) survivors remaining on long-term dialysis, so far only four patients have received a kidney transplant. CONCLUSION: Postoperative dialysis affected long-term mortality. In 75% of survivors renal function improved, but still the majority of patients had an impaired renal function (CKD stage 3-5) at end of follow-up. Future studies should elucidate the impact of kidney dysfunction and dialysis on recipients' long-term survival.

Fewer cytomegalovirus complications after kidney transplantation by de novo use of mTOR inhibitors in comparison to mycophenolic acid.

BACKGROUND: Cytomegalovirus (CMV) is a risk factor for patient and graft survival after kidney transplantation. METHODS: We retrospectively analyzed risk factors for CMV infection in 348 patients who received a kidney transplant donated after brain death (n = 232) or by living donation (n = 116) between 2008 and 2013. Of the 348 patients analyzed, 91 received a mammalian target of rapamycin inhibitor (mTORi)-based immunosuppressive regimen. A total of 266 patients were treated with standard immunosuppression (Group 1) consisting of basiliximab induction, calcineurin inhibitor (CNI), and either mycophenolic acid (MPA, n = 219) or everolimus (EVE) (n = 47). We also included 82 patients who received more intense immunosuppression (Group 2) with lymphocyte depletion, CNI, plus either MPA (n = 38) or EVE (n = 44). Only patients in the high-risk constellation received CMV prophylaxis in Group 1, while all patients in Group 2 received prophylaxis for 6 month. RESULTS: The overall rate of CMV infections was low with 10.1% in all patients. Despite the different prophylaxis strategies applied, no difference was seen in CMV infections between Group 1 (10.9%) and Group 2 (13.6%). A multivariate analysis revealed that patients on EVE had fewer CMV complications compared with patients on MPA (P = 0.013, odds ratio [OR] 4.8, confidence interval [CI] 1.4-16.5). Donor and recipient age >65 years was an independent risk factor (P = 0.002, OR 3.2, CI 1.5-6.7) for CMV infections. Patients with CMV infections had significantly worse graft function after 2 years (P = 0.001). CONCLUSION: CMV is a significant risk factor for long-term graft outcome. Patients treated with EVE developed fewer CMV complications compared to patients on MPA. The use of mTORi is useful in patients at high risk of developing CMV infections.

Two patients with history of STEC-HUS, posttransplant recurrence and complement gene mutations.

Hemolytic uremic syndrome (HUS) is a disease of microangiopathic hemolytic anemia, thrombocytopenia and acute renal failure. About 90% of cases are secondary to infections by Escherichia coli strains producing Shiga-like toxins (STEC-HUS), while 10% are associated with mutations in genes encoding proteins of complement system (aHUS). We describe two patients with a clinical history of STEC-HUS, who developed end-stage renal disease (ESRD) soon after disease onset. They received a kidney transplant but lost the graft for HUS recurrence, a complication more commonly observed in aHUS. Before planning a second renal transplantation, the two patients underwent genetic screening for aHUS-associated mutations that revealed the presence of a heterozygous CFI mutation in patient #1 and a heterozygous MCP mutation in patient #2, and also in her mother who donated the kidney. This finding argues that the two cases originally diagnosed as STEC-HUS had indeed aHUS triggered by STEC infection on a genetic background of impaired complement regulation. Complement gene sequencing should be performed before kidney transplantation in patients who developed ESRD following STEC-HUS since they may be undiagnosed cases of aHUS, at risk of posttransplant recurrence. Furthermore, genetic analysis of donors is mandatory before living-related transplantation to exclude carriers of HUS-predisposing mutations.

The effect of mTOR-inhibition on NF-κB activity in kidney ischemia-reperfusion injury in mice.

Kidney ischemia-reperfusion injury (IRI) is associated with a robust inflammatory response, which is regulated by nuclear factor-kappaB (NF-κB), mainly its heterodimeric form p65/p50. Considering immunomodulatory properties of mammalian target of rapamycin (mTOR) inhibitors, the effect of everolimus on NF-κB activation in kidney IRI was determined in this study. IRI was induced in C57/BL6 mice by clamping both renal pedicles for 45 minutes. Application of everolimus (0.25 mg/kg bw subcutaneously daily) was started one day before IRI induction. Both everolimus-treated and nontreated mice were sacrificed at several times starting at 30 minutes and finishing on day 7 after IRI induction. The NF-κB activity, proinflammatory cytokines IL-1ß, TNF-α, and anti-inflammatory cytokine IL-10 production were determined in kidneys. Compared with nontreated animals, everolimus-treated animals showed significantly increased TNF-α (2741.6 ± 201.72 pg/mg; 1925 ± 185.81 pg/mg, P < .05) and IL-1ß (11.47 ± 1.2 pg/mg; 4.3 ± 0.13 pg/mg, P < .01) production on day 2 after IRI induction accompanied by significantly greater NF-κB/DNA binding activity and p65 nuclear expression (P < .01). Two hours after IRI induction, everolimus-treated animals showed significantly increased IL-1ß mRNA expression (P < .05) followed by increased IL-1ß protein concentrations when compared with nontreated animals measured 6 hours after IRI induction (11.71 ± 1.5 pg/mg; 7.5 ± 1.11 pg/mg, P < .01). Both experimental groups showed increased NF-κB/DNA binding activity at 7 days after IRI induction. Significantly increased nuclear p65 expression was measured in nontreated animals (P < .01), whereas everolimus-treated hosts showed significantly increased nuclear RelB expression (P < .01). These data suggested that everolimus potentiated innate immunity in the early phase of IRI, stimulating the production of NF-κB-driven proinflammatory cytokines such as TNF-α and IL-1ß. The NF-κB activity was potentiated under m-TOR inhibition during kidney IRI, implicating a possible beneficial role of alternative NF-κB activation during the repair phase.

[Long-term outcome with end-stage renal disease - survival is not enough: does dialysis or kidney transplantation matter?]. / Langzeitüberleben bei chronischer Niereninsuffizienz.

Patients with end-stage renal disease require renal replacement therapy with either dialysis or kidney transplantation. Survival and quality of life (QoL) after transplantation are superior to chronic dialysis. Early living donor kidney transplantation is best for patient and graft survival. Preemptive living-related kidney transplantation therefore is the best medical treatment option for these patients. Patients with end-stage renal disease suffer from multiple physical and psychological complaints. The prevalence of depressive disorders is 20-25% in this population. Studies on QoL in children after kidney transplantation show a reduced physical QoL, but an overall good psychological QoL. Alarming results of numerous studies are the high non-adherence rates in adolescents. Especially exercise interventions during dialysis and after kidney transplantation show promising results. Whether QoL of patients will improve with new approaches to immunosuppressive therapy remains to be evaluated in future studies.

A new mouse model of immune-mediated podocyte injury.

Podocytes play a major role in the initiation and progression of glomerular diseases and are a target of both immune-mediated and non-immune-mediated injury. To establish a mouse model of such injury, we preimmunized mice with Freunds adjuvant 5 days before intravenous injection of a rabbit polyclonal antibody directed against a murine podocyte cell line. For the next 7 weeks, we collected urine, serum, and kidney samples. Nephritic animals developed severe albuminuria, which was maximal on day 10. Histochemistry revealed diffuse mesangial matrix expansion. Mouse immunoglobulin G and complement were detected in a linear pattern along the glomerular filtration barrier and in the mesangial hinge region. Complement depletion, however, did not prevent proteinuria. Glomerular T cells were increased, whereas podocytes were significantly reduced. Glomerular foot processes were flattened in regions with mesangial matrix deposition as viewed by electron microscopy. Immunohistochemistry detected the injected anti-podocyte antibody exclusively at the glomerular tuft on all days examined. Immunoelectron microscopy localized the antibody to podocyte foot processes and the glomerular basement membrane, which was morphologically intact. This suggests that the podocyte was the main target of the antiserum. Our study establishes a new mouse model of immune-mediated podocyte injury.

High glucose induces type 1 hexokinase gene expression in isolated glomeruli of diabetic rats and in mesangial cells.

BACKGROUND: Diabetic nephropathy, which is characterized by renal hypertrophy and accumulation of extracellular matrix, is one of the leading causes for end-stage renal disease. Pathophysiological changes, which finally lead to the development of diabetic nephropathy, act through an increase in the intracellular NADH/NAD ratio and the activation of the polyol and protein kinase C pathways. The first rate-limiting enzymes in intracellular glucose metabolism are the hexokinases, which catalyze the phosphorylation of glucose. Therefore, in order to examine a possible link between increased glucose metabolism and the development of diabetic nephropathy mRNA and protein expression as well as enzyme activity of type 1 hexokinase were examined in kidneys of control and diabetic rats and in mesangial cells. METHODS: Diabetes in rats was induced by intravenous injection of streptozotocin and animals were treated or not treated with insulin. RNA or protein was extracted from isolated glomeruli at different time intervals. In addition, glomerular mesangial cells were incubated in high glucose medium and hexokinase expression determined along with enzyme activity. RESULTS: The experiments demonstrate a significant increase in gene and protein expression of type 1 hexokinase in glomeruli of diabetic rats throughout a three week observation period. Insulin therapy reduced glomerular type 1 hexokinase mRNA expression. Gene expression and hexokinase enzyme activity were also increased in mesangial cells grown in high glucose medium. CONCLUSION: The present experiments demonstrate that the expression of type 1 hexokinase is increased in isolated glomeruli of diabetic animals and is regulated by high ambient glucose concentrations. These results add further evidence to the fact that the kidney is one of the tissues most sensitive to high glucose levels in diabetes.

Renal handling of human apolipoprotein(a) and its fragments in the rat.

The sites and mechanisms of the catabolism of atherogenic lipoprotein(a) (Lp(a)) are not well understood. Lp(a) is increased in patients with end-stage renal disease, suggesting a renal catabolism of Lp(a). To gain a better insight into renal handling of Lp(a), we established a heterologous rat model to study the renal catabolism of human Lp(a). Pure human Lp(a) was injected into Wistar rats, and animals were sacrificed at different time points (30 minutes to 24 hours). Intact Lp(a) was cleared from the circulation of injected rats with a half-life time of 14.5 hours. Strong intracellular immunostaining for apolipoprotein(a) (apo(a)) was observed in the cytoplasm of proximal tubular cells after 4, 8, and 24 hours. Apolipoprotein B (apoB) was colocalized with glomerular apo(a) 1 to 8 hours after Lp(a) injection, but renal capillaries and tubules remained negative. No relevant amounts of apo(a) fragments were found in the plasma of rats after injection of Lp(a). During all urine collection periods, apo(a) fragments with molecular weights of 50 to 160 kd were detected in the urine, however. Our results show that human Lp(a) injected into rats accumulates intracellularly in the rat kidney, and apo(a) fragments are excreted in the urine. The kidney apparently plays a major role in fragmentation of Lp(a). Despite the fact that rodents lack endogenous Lp(a), rats injected with human Lp(a) may provide a useful heterologous animal model to study the renal metabolism of Lp(a) further.

Monocyte chemoattractant protein-1 and osteopontin differentially regulate monocytes recruitment in experimental glomerulonephritis.

BACKGROUND: This study evaluated the mechanisms of monocyte/macrophage (M/M) infiltration in a rat model of anti-glomerular basement membrane glomerulonephritis (GN). We focused on chemokines and osteopontin, which are known regulators of M/M recruitment. METHODS: Using immunohistology, in situ hybridization, and Northern blotting, the expression levels of chemokines and osteopontin were evaluated in isolated glomeruli and tubules 4, 10, and 20 days after the induction of GN. In vivo blocking experiments were performed by application of neutralizing antibodies against osteopontin and monocyte chemoattractant protein-1 (MCP-1). RESULTS: In nephritic animals, high glomerular MCP-1 and RANTES (regulated upon activation normal T cell expressed and secreted) expression levels were observed on days 4 and 10. The tubular expression of MCP-1, however, was only slightly enhanced. In contrast, tubular osteopontin production was maximally stimulated (day 10) and paralleled with peaks of albuminuria and tubulointerstitial M/M infiltration. Application of an anti-osteopontin antibody ameliorated tubulointerstitial and glomerular M/M recruitment, whereas treatment with an anti-MCP-1 antibody selectively reduced glomerular M/M recruitment. However, tubulointerstitial M/M infiltration remained unchanged. CONCLUSION: These studies show that chemokines and osteopontin are differentially expressed in glomeruli and tubules in this model of GN. Chemokines play a primary role in the glomeruli, whereas osteopontin has a predominant role in tubulointerstitial M/M recruitment. The roles of chemokines and osteopontin may thus be dependent on the renal compartment and on the disease model.

Mechanisms underlying endothelial dysfunction in diabetes mellitus.

Incubation of endothelial cells in vitro with high concentrations of glucose activates protein kinase C (PKC) and increases nitric oxide synthase (NOS III) gene expression as well as superoxide production. The underlying mechanisms remain unknown. To address this issue in an in vivo model, diabetes was induced with streptozotocin in rats. Streptozotocin treatment led to endothelial dysfunction and increased vascular superoxide production, as assessed by lucigenin- and coelenterazine-derived chemiluminescence. The bioavailability of vascular nitric oxide (as measured by electron spin resonance) was reduced in diabetic aortas, although expression of endothelial NOS III (mRNA and protein) was markedly increased. NOS inhibition with N:(G)-nitro-L-arginine increased superoxide levels in control vessels but reduced them in diabetic vessels, identifying NOS as a superoxide source. Similarly, we found an activation of the NADPH oxidase and a 7-fold increase in gp91(phox) mRNA in diabetic vessels. In vitro PKC inhibition with chelerythrine reduced vascular superoxide in diabetic vessels, whereas it had no effect on superoxide levels in normal vessels. In vivo PKC inhibition with N:-benzoyl-staurosporine did not affect glucose levels in diabetic rats but prevented NOS III gene upregulation and NOS-mediated superoxide production, thereby restoring vascular nitric oxide bioavailability and endothelial function. The reduction of superoxide in vitro by chelerythrine and the normalization of NOS III gene expression and reduction of superoxide in vivo by N:-benzoyl-staurosporine point to a decisive role of PKC in mediating these phenomena and suggest a therapeutic potential of PKC inhibitors in the prevention or treatment of vascular complications of diabetes mellitus. The full text of this article is available at http://www.circresaha.org.

Renovascular hypertension does not influence repair of glomerular lesions induced by anti-thymocyte glomerulonephritis.

BACKGROUND: Systemic hypertension is a risk factor for progression of renal disease. However, it is not clear whether hypertension has an effect on healing or regression of immune-mediated glomerular damage. To evaluate this effect, we applied a model of glomerulonephritis in rats with two-kidney, one-clip hypertension and studied the effect of hypertension on the healing process of this nephritis. METHODS: The anti-thymocyte serum (ATS) glomerulonephritis was induced in rats six weeks after initiation of two-kidney, one-clip hypertension, when blood pressure was already increased. Renal structure and function were examined six weeks later. Glomerular expression of alpha smooth muscle actin, the cell cycle inhibitor p27Kip1, and transforming growth factor-beta (TGF-beta) was evaluated by Western blotting. Glomerular proliferation, monocyte infiltration, and fibronectin were examined by immunohistochemistry. RESULTS: Decreased survival, an increase of proteinuria, as well as increased glomerular and tubulointerstitial damage, were found in hypertensive rats compared with normotensive rats. Expression of fibronectin, alpha-smooth muscle actin, TGF-beta, and p27Kip1 was increased in the nonclipped kidney. Complete healing of the glomerular changes associated with the nephritis occurred in normotensive nephritic rats. Surprisingly, complete healing of the nephritis was also found in the clipped as well as nonclipped kidneys of renovascular hypertensive rats. No significant differences could be found for survival, proteinuria, glomerular size, proliferation, monocyte/macrophage infiltration, sclerosis, tubulointerstitial damage, as well as expression of alpha-smooth muscle actin, TGF-beta, fibronectin, and p27Kip1 between hypertensive rats with and without nephritis. CONCLUSION: These data demonstrate that renovascular hypertension does not influence healing of the glomerular lesions in the anti-thymocyte serum nephritis. This is a rather surprising observation and leaves the question open of which role, in fact, blood pressure may have on the reparative phase of an acute glomerulonephritis, or whether its role depends on the type of glomerulonephritis.

Effect of renovascular hypertension on experimental glomerulonephritis in rats.

Systemic hypertension is a major risk factor that determines the rate of progression of kidney disease. The underlying mechanisms, however, are incompletely understood. To gain insight into these mechanisms, the present study was undertaken to characterize the effects of renovascular hypertension on the course of anti-thymocyte antibody-induced glomerulonephritis. Glomerulonephritis was induced in rats 6 weeks after the initiation of two-kidney, one-clip hypertension, when blood pressure was already increased. Structure and function of the clipped and the nonclipped kidney were examined 5 days later. Glomerular filtration rate (GFR) was measured by inulin clearance. The induction of nephritis did not alter the blood pressure in either hypertensive rats or normotensive controls. Albuminuria increased slightly in normotensive rats after the induction of nephritis, whereas no significant differences were found between hypertensive rats with or without nephritis. No significant differences were found for the GFR values of normotensive controls and nephritic animals or for values in the clipped kidney with or without nephritis. However, the GFR of the nonclipped kidney was significantly reduced in nephritic animals as compared with all other groups. Morphologic evaluation revealed that hypertensive rats with nephritis exhibited a combination of characteristics of nephritis and hypertensive glomerular injury. Histologic findings of nephritis, such as glomerular binding of rabbit IgG and glomerular proliferation and mesangial matrix expansion, were similar after the induction of nephritis in controls and in the clipped and nonclipped kidneys of hypertensive animals. However, intraglomerular microaneurysms were significantly more often found in the non-clipped kidneys after the induction of nephritis. Hypertension-induced deterioration of glomerular function was not associated with marked morphologic deterioration but rather with a combination of the characteristics of nephritis and hypertensive glomerular injury.

Leptin stimulates proliferation and TGF-beta expression in renal glomerular endothelial cells: potential role in glomerulosclerosis [seecomments].

BACKGROUND: Leptin inhibits food intake and increases energy expenditure. Although the kidney expresses abundant transcripts of the short form of the leptin receptor (Ob-Ra), a role for this hormone in renal function remains unclear. Because individuals with massive obesity who may exhibit increased leptin serum concentrations develop renal glomerulosclerosis, we studied whether leptin can influence renal growth and profibrogenic processes. METHODS: The effects of recombinant leptin on proliferation and synthesis of transforming growth factor-beta1 (TGF-beta1) was investigated in cultured glomerular endothelial cells of the rat (GERs) and syngeneic mesangial cells. Furthermore, leptin receptor expression and potential signal transduction pathways were evaluated in GERs. In addition, leptin was also infused for different time periods (72 hr and 3 weeks) into naive rats. RESULTS: Recombinant mouse leptin induced proliferation of GERs, but not of syngeneic mesangial cells. Coincubation with angiotensin II and leptin exerts additive proliferative effects in GERs. An antileptin-receptor antibody totally abolished this proliferation but did not influence serum-induced proliferation. GER expressed high affinity receptors of the Ob-Ra type (Kd, 4 nM; Bmax, 9700 receptors/cell). Leptin also stimulated phosphorylation of STAT1alpha, and kinase inhibitors attenuated proliferation, suggesting a pivotal role of phosphorylation in this process. Incubation of GERs with leptin also induced mRNA expression of TGF-beta1 and enhanced secretion of this profibrogenic cytokine. Short-term leptin infusion (72 hr) into naive rats induced a significant proliferation, mainly restricted to glomerular endothelial cells, and enhanced glomerular TGF-beta1 mRNA levels. In rats continuously infused for three weeks with leptin, glomerular TGF-beta1 expression was still enhanced, and an additional increase in glomerular collagen type IV mRNA and protein expression was detected. These animals revealed an increase in proteinuria compared with control-infused rats. CONCLUSION: Our findings are the first in vitro and in vivo demonstration that leptin is a renal growth and profibrogenic factor. These results may be an important contribution to our understanding of how leptin can contribute to renal damage, characterized by endocapillary proliferation and subsequent development of glomerulosclerosis, in pathophysiological situations with high circulating levels such as in diabetics or obese individuals. Although the effects of leptin itself are moderate, growth-promoting and profibrogenic effects may be enhanced in concert with other factors such as angiotensin II.

Monocyte chemoattractant protein-1 mediates collagen deposition in experimental glomerulonephritis by transforming growth factor-beta.

BACKGROUND: Monocyte chemoattractant protein-1 (MCP-1) plays a significant role in the recruitment of monocytes/macrophages in experimental glomerulonephritis (GN). Because recent evidence points to possible profibrogenic effects of leukocyte-derived factors in GN, this study was designed to evaluate the role of the chemokine MCP-1 in the fibrogenesis of experimental GN. METHODS: Rats with an anti-thy-1-induced GN were treated with a neutralizing antiserum against MCP-1. Glomerular collagen type IV, as a marker of glomerular matrix deposition, was assessed by Northern and Western blotting and immunohistology. Transforming growth factor-beta (TGF-beta), an important mediator of this matrix expansion, was studied by Northern and Western blotting. RESULTS: The induction of GN resulted in a significant increase of glomerular collagen type IV deposition and TGF-beta synthesis. The neutralization of MCP-1 significantly reduced the enhanced collagen type IV protein synthesis and deposition without affecting collagen mRNA expression. However, both the enhanced transcription and protein synthesis of TGF-beta were inhibited by anti-MCP-1 antiserum in nephritic animals. CONCLUSIONS: In this model of GN, MCP-1 has a fibrogenic effect through the stimulation of TGF-beta. MCP-1 is thus not only important for the recruitment of inflammatory cells, but also mediates glomerular matrix accumulation.

Cyclooxygenase metabolites mediate glomerular monocyte chemoattractant protein-1 formation and monocyte recruitment in experimental glomerulonephritis.

BACKGROUND: Monocyte chemoattractant protein-1 (MCP-1) has been shown to play a significant role in the recruitment of monocytes/macrophages in experimental glomerulonephritis. Whereas a number of inflammatory mediators have been characterized that are involved in the expression of MCP-1 in renal disease, little is known about repressors of chemokine formation in vivo. We hypothesized that cyclooxygenase (COX) products influence the formation of MCP-1 and affect inflammatory cell recruitment in glomerulonephritis. METHODS: The effect of COX inhibitors was evaluated in the antithymocyte antibody model and an anti-glomerular basement membrane model of glomerulonephritis. Rats were treated with the COX-1/COX-2 inhibitor indomethacin and the selective COX-2 inhibitors meloxicam and SC 58125. Animals were studied at 1 hour, 24 hours, and 5 days after induction of the disease. RESULTS: Indomethacin, to a lesser degree the selective COX-2 inhibitors, enhanced glomerular MCP-1 and RANTES mRNA levels. Indomethacin enhanced glomerular monocyte chemoattractant activity an the infiltration of monocytes/macrophages at 24 hours and 5 days. CONCLUSIONS: Our studies demonstrate that COX products may serve as endogenous repressors of MCP-1 formation in experimental glomerulonephritis. The data suggest that COX-1 and COX-2 products mediate these effects differently because the selective COX-2 inhibitors had less influence on chemokine expression.

L-arginine suppresses lipopolysaccharide-induced expression of RANTES in glomeruli.

Endotoxemia leads to the infiltration of inflammatory cells in glomeruli and the tubulointerstitium of the kidney. The ultimate mechanisms for this infiltration, however, are not entirely clear. In this study, the glomerular formation of the chemokine RANTES (regulated upon activation normal T cell expressed and secreted) was examined in an in vivo model of endotoxemia to evaluate the role the local release of chemokines might play in the regulation of this inflammatory cell infiltrate. Since the beneficial effects of nitric oxide (NO) on immune-mediated tissue injury have been reported, we also examined possible interactions between the chemokine RANTES and the L-arginine/NO pathway. To induce endotoxemia, rats were injected intraperitoneally with lipopolysaccharide (LPS). Glomeruli were isolated over a 24-h time period, and RANTES was assessed by Northern blotting, a chemotactic assay, and a specific enzyme-linked immunosorbent assay. The chemokine release was associated with increased glomerular infiltration of monocytes/macrophages. LPS also stimulated the mRNA expression of inducible NO synthase and increased the release of nitrite into the supernatants of isolated glomeruli. Supplementation of L-arginine intake increased the release of glomerular nitrite and reduced glomerular RANTES expression after the injection of LPS. Inhibition of the L-arginine/NO pathway by the unspecific NO synthase inhibitor N(G)-nitro-L-arginine methylester significantly increased glomerular RANTES mRNA expression and the number of infiltrating glomerular macrophages. These data demonstrate that L-arginine suppresses glomerular RANTES formation and suggest that the chemokine-mediated recruitment of glomerular macrophages in LPS-induced endotoxemia can be modulated by the L-arginine/NO pathway.

Glomerular expression of p27Kip1 in diabetic db/db mouse: role of hyperglycemia.

Early diabetic nephropathy is characterized by glomerular hypertrophy. Previous studies in vitro have demonstrated that mesangial cells exposed to high glucose are arrested in the G1-phase of the cell cycle and express increased levels of the cyclin-dependent kinase inhibitor p27Kip1. The present study was performed to investigate the renal expression of p27Kip1 in db/db mice, a model of diabetes mellitus type II. Glomerular p27Kip1 protein, but not mRNA expression, was strongly enhanced in diabetic db/db mice compared with non-diabetic db/+ littermates. Immunohistochemical studies revealed that this stimulated expression was mainly restricted to the nuclei of mesangial cells and podocytes, but glomerular endothelial cells occasionally also stained positively. Quantification of p27Kip1 positive glomerular cells showed a significant increase of these cells in db/db mice compared with non-diabetic db/+ animals. Although tubular cells revealed a positive staining for p27Kip1 protein, there was no difference between db/+ and db/db mice. Immunoprecipitation experiments revealed that p27Kip1 protein associates with Cdk2 and Cdk4, but not with Cdk6. To test for the influence of hyperglycemia on cell cycle arrest and p27Kip1 expression, mesangial cells were isolated from db/+ and db/db mice. There was a similar basal proliferation when these cells were grown in normal glucose-containing medium (100 mg/dl). However, raising the glucose concentration to 275 to 450 mg/dl induced cell cycle arrest in db/+ as well as db/db mesangial cells. Increasing the medium osmolarity with D-mannitol failed to induce p27Kip1 expression in mesangial cells. Transfection of cells with p27Kip1 antisense, but not missense, phosphorothioate oligonucleotides facilitated cell cycle progression equally well in db/+ and db/db mesangial cells. Furthermore, p27Kip1 expression was comparable in both cell lines in normal glucose, but increased in high glucose medium. Our studies demonstrate that p27Kip1 expression is enhanced in diabetic db/db animals. This induction appears to be due to hyperglycemia. Expression of p27Kip1 may be important in cell cycle arrest and hypertrophy of mesangial cells during early diabetic nephropathy.

High glucose stimulates expression of p27Kip1 in cultured mouse mesangial cells: relationship to hypertrophy.

Hypertrophy of mesangial cells is an early hallmark of diabetic nephropathy. We have previously shown that murine mesangial cells (MMC), cultured in high-glucose medium, are arrested in the G1 phase of the cell cycle and undergo hypertrophy. This study was undertaken to test whether high glucose-containing medium influences the expression of p27Kip1, an inhibitor of G1 phase active cyclin-dependent kinases (CDK). Incubation of MMC, in the absence of other factors for 48-96 h, in medium containing high D-glucose (450 mg/dl), stimulated p27Kip1 protein expression but failed to influence mRNA abundance. These effects were independent of the osmolarity of the medium. High glucose-stimulated expression of p27Kip1 involved activation of protein kinase C and was partly dependent on induction of transforming growth factor-beta (TGF-beta). Immunoprecipitation experiments revealed that only small amounts of p27Kip1 protein from MMC grown in high-glucose medium preferentially associates with CDK2 but not with CDK4. The p27Kip1 antisense, but not missense, oligonucleotides inhibited high glucose-stimulated total protein synthesis and facilitated G1 phase exit. Our data showed for the first time that expression of p27Kip1 protein is pivotal in mesangial cell hypertrophy induced by high ambient glucose. These findings may be important in the deciphering of molecular processes causing diabetic glomerular hypertrophy.

Angiotensin II stimulates expression of the chemokine RANTES in rat glomerular endothelial cells. Role of the angiotensin type 2 receptor.

Glomerular influx of monocytes/macrophages (M/M) occurs in many immune- and non-immune-mediated renal diseases. The mechanisms targeting M/M into the glomerulus are incompletely understood, but may involve stimulated expression of chemokines. We investigated whether angiotensin II (ANG II) induces the chemokine RANTES in cultured glomerular endothelial cells of the rat and in vivo. ANG II stimulated mRNA and protein expression of RANTES in cultured glomerular endothelial cells. The ANG II-induced RANTES protein was chemotactic for human monocytes. Surprisingly, the ANG II-stimulated RANTES expression was transduced by AT2 receptors because the AT2 receptor antagonists PD 123177 and CGP-42112A, but not an AT1 receptor blocker, abolished the induced RANTES synthesis. Intraperitoneal infusion of ANG II (500 ng/h) into naive rats for 4 d significantly stimulated glomerular RANTES mRNA and protein expression compared with solvent-infused controls. Immunohistochemistry revealed induction of RANTES protein mainly in glomerular endothelial cells and small capillaries. Moreover, ANG II- infused animals exhibited an increase in glomerular ED-1- positive cells compared with controls. Oral treatment with PD 123177 (50 mg/liter drinking water) attenuated the glomerular M/M influx without normalizing the slightly elevated systolic blood pressure caused by ANG II infusion, suggesting that the effects on blood pressure and RANTES induction can be separated. We conclude that the vasoactive peptide ANG II may play an important role in glomerular chemotaxis of M/M through local induction of the chemokine RANTES. The observation that the ANG II- mediated induction of RANTES is transduced by AT2 receptors may influence the decision as to which substances might be used for the therapeutic interference with the activity of the renin-angiotensin system.