Urinary Cytokine Excretion Research Articles

Relationships between inflammation, hemodynamic function and RAAS in longstanding type 1 diabetes and diabetic kidney disease.

The renin angiotensin aldosterone system (RAAS) is associated with renal disease and inflammation in a diabetes setting, however, little is known about the implicated mechanisms in individuals with long standing diabetes. Accordingly, our aim was to perform an observational study to quantify urinary excretion of inflammatory biomarkers in participants with long standing type 1 diabetes (T1D) (with and without diabetic kidney disease [DKD]) and controls, at baseline and in response to RAAS activation. GFRINULIN, ERPFPAH, and 42 urine inflammatory biomarkers were measured in 74 participants with T1D for ≥50 years (21 with DKD and 44 without DKD [DKD resistors]) and 73 healthy controls. Additionally, inflammatory biomarkers were measured before and after an angiotensin II infusion (ANGII, 1 ng∙kg-1∙min-1). Significantly lower urinary excretion of cytokines (IL-18, IL-1RA, IL-8), chemokines (MCP1, RANTES) and growth factors (TGF-α, PDGFAA, PDGFBB, VEGF-A) was observed in participants with T1D at baseline compared to controls. Urinary IL-6 was higher in DKD than in DKD resistors in an exploratory analysis unadjusted for multiple comparisons. In T1D only, lower GFRINULIN correlated with greater excretion of proinflammatory biomarkers (IL-18, IP-10, & RANTES), growth factors (PDGF-AA & VEGFAA), and chemokines (eotaxin & MCP-1). ANGII increased 31 of 42 inflammatory biomarkers in T1D vs controls (p < 0.05), regardless of DKD resistor status. In conclusion, lower GFR and intra-renal RAAS activation were associated with increased inflammation even after longstanding T1D. The increased urinary IL-6 in patients with DKD requires further investigation to determine whether IL-6 is a candidate protective biomarker for prognostication or targeted therapy in DKD.

Renal Inflammation in DOCA-Salt Hypertension.

Recent reports indicate that, in addition to treating hypertension, renal denervation (RDN) also mitigates renal inflammation. However, because RDN decreases renal perfusion pressure, it is unclear whether these effects are because of the direct effects of RDN on inflammatory signaling or secondary to decreased arterial pressure (AP). Therefore, this study was conducted to elucidate the contribution of renal nerves to renal inflammation in the deoxycorticosterone (DOCA)-salt rat, a model in which RDN decreases AP and abolishes renal inflammation. In Experiment 1, we assessed the temporal changes in renal inflammation by measuring renal cytokines and AP in DOCA-salt rats. Uninephrectomized (1K) adult male Sprague Dawley rats that received surgical RDN or sham (Sham) were administered DOCA (100 mg, SC) and 0.9% saline for 21 days. AP was measured by radiotelemetry, and urinary cytokine excretion was measured repeatedly. In Experiment 2, the contribution of renal nerves in renal inflammation was assessed in a 2-kidney DOCA-salt rat to control for renal perfusion pressure. DOCA-salt treatment was administered after unilateral (U-)RDN. In Experiment 1, DOCA-salt-induced increases in AP and renal inflammation (assessed by urinary cytokines) were attenuated by RDN versus Sham. In Experiment 2, GRO/KC (growth-related oncogene/keratinocyte chemoattractant), MCP (monocyte chemoattractant protein)-1, and macrophage infiltration were lower in the denervated kidney versus the contralateral Sham kidney. No differences in T-cell infiltration were observed. Together, these data support the hypothesis that renal nerves mediate, in part, the development of renal inflammation in the DOCA-salt rat independent of hypertension. The mechanisms and cell-specificity mediating these effects require further investigation.

RENAL SYMPATHETIC NERVES AND INFLAMMATION IN HYPERTENSION

Objective: Renal denervation (RDN) may be an effective treatment for hypertension; however, the mechanisms remain unknown. RDN mitigates hypertension in addition to the concomitant renal inflammation in the deoxycorticosterone (DOCA)-salt rat model. Renal inflammation is hypothesized to drive the hypertension, but the timing and relationship to mean arterial pressure (MAP) remain elusive. We aimed to elucidate the temporal inflammatory and MAP response to DOCA-salt through urinary cytokine excretion, and test its responsiveness to renal denervation. We hypothesized that (a) RDN would mitigate renal and urinary cytokine content, and (b) a decrease urinary cytokine would precede changes in MAP. Design and method: 12 uninephrectomized male Sprague Dawley rats (300–325 g) received either surgical renal denervation (RDN; n = 6) or sham (Sham; n = 6). One week following, rats were administered DOCA (100 mg) and 0.9% saline for 21 days. Urine samples were collected weekly. Urinary and renal tissue cytokines (IL-1β, IL-2, IL-6, GRO/KC, MCP-1) were measured by multiplex assay. Temporal data analyzed by two-way ANOVA with Bonferroni post-hoc test (α=0.05). All other data were analyzed by Student's t-test. Data presented as mean ± SEM. Results: MAP response to DOCA-salt was attenuated (*p < .05) by RDN vs. Sham, where MAP significantly diverged at Days 14–21 (see Table). Similarly, all measured renal cytokines were markedly reduced (*p < .05) in RDN vs. Sham - a reduction of 42–70% of Sham values (see Table). Urinary cytokine excretion on day 21 mirrored this effect, where each cytokine was lower (*p < .05) in RDN vs. Sham – reduced by 44–84% Sham values (see Table). No difference was detected between RDN and Sham urinary cytokine content on Days 0–7, and only urinary IL-1β and IL-2 were reduced (*p < .05) at Day 14.Conclusions: In conclusion, these data support our hypothesis that urinary cytokines reflect renal inflammatory cytokine content, and both are abated by RDN. However, our second hypothesis was not supported, as differences in MAP appeared prior to a significant reduction of most urinary cytokines. With these findings, we hypothesize that hypertension may precede renal inflammation in this model. Further studies are necessary to elucidate the driving force for renal inflammation in this model.

Renal Sympathetic Nerves and Inflammation in Hypertension: Assessing Temporal Renal Inflammation Responses to Renal Denervation by Urinary Cytokine Excretion in the DOCA‐Salt Rat

Preclinical and clinical studies demonstrate renal sympathetic denervation (RDN) may be an effective treatment for hypertension and end stage renal disease; however, the mechanisms of this effect remain unknown. We recently reported (RDN) mitigates hypertension in addition to the concomitant renal inflammation in the deoxycorticosterone acetate (DOCA)‐salt rat model. In this model, renal inflammation is hypothesized to drive the hypertension, but the timing and relationship to arterial pressure remain elusive. In this study, we aimed to elucidate the temporal renal inflammatory and arterial pressure response to DOCA‐salt through repeated‐measurement of urinary pro‐inflammatory cytokine excretion, and test the responsiveness to renal sympathetic denervation. We hypothesize that (a) RDN would mitigate renal and urinary cytokine content, and (b) a decrease urinary cytokine content due to RDN would precede changes in arterial pressure. To test this hypothesis, 12 uninephrectomized adult male Sprague Dawley rats (300–325 g) were implanted with radiotelemeters to measure 24‐hour mean arterial pressure (MAP) weekly, and received either surgical renal denervation (RDN; n=6) or sham procedure (Sham; n=6). One week following, both groups were administered DOCA (100 mg, s.c.) and 0.9% saline for 21 days. 6‐hour urine samples were collected weekly over ice using metabolic cage housing. At day 21, rats were anesthetized, and renal tissue was collected for cytokine and histological analysis. Pro‐inflammatory cytokines (IL‐1β, IL‐2, IL‐6, GRO/KC, MCP‐1) were measured by Luminex assay in both urine and renal tissue. Renal and urinary cytokine content were normalized to total protein and creatinine, respectively. Temporal response data analyzed by two‐way repeated measured ANOVA with Bonferroni post‐hoc test (α=0.05). All other data were analyzed by Student's t‐test. Data presented as mean±SEM. MAP response over 21‐day DOCA‐salt treatment was attenuated (*p&lt;.05) by RDN vs. Sham, where MAP significantly diverged at Days 14–21 (see Data Table). Similarly, all measured pro‐inflammatory cytokines in renal parenchymal protein isolates were markedly reduced (*p&lt;.05) in RDN vs. Sham ‐ a reduction of 42–70% of Sham values (see Data Table). Interestingly, urinary cytokine excretion on day 21 mirrored this effect, where each cytokine was lower (*p&lt;.05) in RDN vs. Sham – similarly reduced by 44–84% Sham values (see Data Table). Of note, no difference was observed between RDN and Sham urinary cytokine content on Days 0–7, and only urinary IL‐1β and IL‐2 were reduced (*p&lt;.05) at Day 14 (see Data Table). Histological analysis of renal injury and immune cell (macrophage, T‐cell) infiltration is currently underway to establish the end organ damage and immune cell specificity in the reported anti‐inflammatory effects of RDN. Taken together, these data support our first hypothesis of urinary cytokines reflect renal inflammatory cytokine content, and both are abated by RDN. However, our second hypothesis was not supported by the current data, as MAP was reduced in the RDN‐treated group prior to a significant reduction of most urinary cytokines. With these findings, we hypothesize that DOCA‐salt hypertension may precede the renal inflammation in this model. Further studies are necessary to elucidate the source of renal inflammation in this model, and its relationship to arterial pressure control.Support or Funding InformationThis project was supported in part by NIH HL116476 (JWO) and AHA 17POST33661003 (CTB).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Impact of Τh1 and Τh2 cytokines in the progression of idiopathic nephrotic syndrome due to focal segmental glomerulosclerosis and minimal change disease.

Background: Differential diagnosis between primary focal segmental glomerulosclerosis (FSGS) and minimal change disease (MCD) is sometimes difficult as nephrotic syndrome is the main clinical symptom in both diseases. Objectives: This study has attempted to evaluate the urinary excretion of Th1 and Th2 cytokines as potential biomarkers in distinguishing the two types of nephrotic syndrome, and predicting outcome of renal function. Patients and Methods: Thirty-six patients with FSGS (M/F 22/14, Age; 41.9 ± 17 years, SCr=1.7 ± 0.8 mg/dL, UProt=4.7 ± 5.5 g/24 h), and 21 with MCD (M/F 5/16, Age; 41.4 ± 15 years, SCr = 1 ± 0.4 mg/dL, UProt = 7.9 ± 9.3 g/24 h) were included in the study. Τh1 (IL-2, IL-12, GM-CSF, INF-γ, TNF-α) and Th2 cytokines (IL-4, IL-5, IL-10, IL-13) were measured by multiple cytokine assay, Luminex technology, in first morning urinary samples collected at the day of renal biopsy. Results: No significant differences in urinary excretion of all cytokines were found between FSGS and MCD patients. In FSGS however, IL-12 urinary levels were independent factor correlated with both global sclerosis (R = 0.5, P = 0.009) and interstitial fibrosis (R = 0.5, P = 0.02). Th1 cytokines (IL-2 and GM-CSF) were significantly increased in FSGS patients who did not respond to treatment (P = 0.03 and P = 0.007, respectively). Th2 cytokines (IL-4, IL-5, IL-10, IL-13) were significantly increased in MCD patients with frequent relapses (P = 0.05, P = 0.001, P = 0.01, P = 0.03). Conclusions: Urinary excretion of Th1 and Th2 cytokines cannot discriminate FSGS from MCD. Th1 cytokines, especially IL-12, IL-2 and GM-CSF, may be involved in pathology and progression of FSGS, while Th2 cytokines are implicated in frequent relapses of nephrotic syndrome in MCD.

T helper (Th)-cytokines in the urine of patients with primary glomerulonephritis treated with immunosuppressive drugs: Can they predict outcome?

BackgroundGlomerulonephritides (GNs) represent common causes of chronic kidney disease associated with a wide spectrum of clinical and histological features. Various factors that activate the inflammatory cascade are involved in the development of kidney injury. The aim of this study was to estimate the urinary excretion of pro-inflammatory (IL-2, INF-γ, TNF-α, IL-6, IL-17) and anti-inflammatory (IL-4, IL-10, TGF-β1) cytokines, as well as the chemokine MCP-1 in patients with various types of GN treated by immunosuppressive drugs and to identify any prognostic value of excreted cytokines for future renal function. Patients and methodsNinety-seven patients (62M/35F, age 53.1±15.6years) with primary glomerulonephritis and 32 healthy controls were studied. The original diagnoses were membranous nephropathy (MN, n=36), IgA nephropathy (IgAN, n=31) and minimal changes disease or focal segmental glomerulosclerosis (MCD/FSGS, n=30). All patients had been treated with immunosuppressive drugs and, at the time of measurement of urinary cytokine excretion, were either in clinical remission or still had active disease with persistent proteinuria. ResultsGN patients had significantly higher levels of all cytokines and MCP-1 compared to healthy controls. A strong positive correlation between TGF-β1 and MCP-1 concentrations was observed in all GN patients. Increased urinary excretion of all tested cytokines apart from TNF-α and TGF-β1 was observed even in patients with clinical remission. The main difference between patients with proteinuria and those in clinical remission was the level of MCP-1 urinary excretion. The urinary excretion of MCP-1 and TGF-β1 was significantly higher in patients with MN who showed deterioration of renal function over a follow-up period of five years. ConclusionsIncreased levels of cytokines are observed in the urine of patients with different types of glomerulonephritis, even after the achievement of clinical remission with the administration of immunosuppressive drugs. Urinary excretion of MCP-1 and TGF-β1 indicates the ongoing inflammatory and fibrotic processes in the kidney and is probably related to unfavourable outcomes.

The effect of garlic powder on human urinary cytokine excretion.

To evaluate the effects of orally administered dehydrated garlic powder on cytokine excretion in the urinary tract. A total of 60 healthy volunteers, randomized into 3 groups, were given a single oral dose of 1 g or 3 g of dehydrated garlic powder or placebo. Urine samples were obtained 6.0 and 24.0 h after garlic intake and assayed for interleukin-8 (IL-8), interleukin- 12 (IL-12), tumor necrosis factor-alpha (TNF-α), diallyl disulfide (DADS) and diallyl sulfide (DAS). Significant increases in IL-12 levels over baseline were noted in urine samples obtained after oral intake of 1 g and 3 g of garlic powder (P < .001). In the 1 g and 3 g garlic powder treatment groups, time-dependent variations in IL-12 levels over the study period were significantly different from the placebo group (P < .001). In both garlic treatment groups, urinary levels of IL-8 and TNF-α were not significantly different from baseline and placebo levels (P > .017). DADS and DAS were not detected in the urine samples at any time after garlic powder intake. Oral intake of doses of garlic traditionally used for daily supplementation increases urinary levels of IL-12, which is a potent stimulator of T helper cell 1 (Th-1) immune responses. This observation encourages further studies investigating the immunostimulatory role of garlic in the urinary tract.

Long-term hemodynamic and molecular effects persist after discontinued renin–angiotensin system blockade in patients with type 1 diabetes mellitus

Animal studies suggest temporary renin-angiotensin system (RAS) blockade enhances long-term vascular protective effects; however, this is not established in humans. Here we evaluated the long-term effects of prior RAS blockade on hemodynamic function, urinary measures of inflammation, and tissue antioxidant mRNA expression in patients with type 1 diabetes mellitus (T1DM) who participated in the 5-year Renin Angiotensin System Study (RASS). At 4 years after completing the RASS and discontinuing study medication, renal hemodynamic responses to clamped hyperglycemia were significantly greater in 18 patients in the RAS blockade group compared to 9 patients of the placebo-treated group. Individuals who had received RAS blockade also exhibited higher flow-mediated vasodilatation, reduced urinary cytokine excretion in response to hyperglycemia, and increased skin mRNA expression of superoxide dismutase-1 and catalase. Thus, patients with uncomplicated T1DM who received prior RAS blockade for 5 years maintain long-term effects on renal hemodynamic and systemic vascular function, inflammatory pathways in the kidney, and antioxidant enzyme expression in skin 4 years after discontinuation of therapy. Our findings suggest that sustained long-term protective effects of finite RAS inhibition requires further study.

Chronic administration of EP4-selective agonist exacerbates albuminuria and fibrosis of the kidney in streptozotocin-induced diabetic mice through IL-6

Diabetic nephropathy is currently the most common cause of end-stage renal disease in the western world. Exacerbated inflammation of the kidney is known to contribute acceleration of nephropathy. Despite increased COX-2-mediated production of prostanoid metabolite PGE2, knowledge on its involvement in the progression of diabetic kidney disease is not complete. Here, we show the cross talk of the PGE2–EP4 pathways and IL-6 in inducing albuminuria and fibrosis in an animal model of type 1 diabetes. Hyperglycemia causes enhanced COX-2 expression and PGE2 production. Administration of PGE2 receptor EP4-selective agonist ONO-AE1-329 for 12 weeks exacerbated fibrosis and albuminuria. Diabetes-induced expression of inflammatory cytokines TNFα and TGFβ1 was enhanced in EP4 agonist-treated mice kidney. In addition, urinary excretion of cytokines (TNFα and IL-6) and chemokines (MCP-1 and IP-10) were significantly more in EP4-treated mice than vehicle-treated diabetes. Diabetes-induced collagen I and CTGF expression were also significantly higher in EP4-treated mice. However, EP4 agonist did not alter macrophage infiltration but increased cytokine and chemokine production in RAW264.7 cells. Interestingly, EP4-induced IL-6 expression in the kidney was localized in proximal and distal tubular epithelial cells. To confirm further whether EP4 agonist increases fibrosis and albuminuria through an increase in IL-6 expression, IL-6-knockout mice were administered with EP4 agonist. IL-6-knockout mice were resistant to EP4-induced exacerbation of albuminuria and diabetes and EP4-induced fibrosis. Our data suggest that EP4 agonist through IL-6 induces glomerulosclerosis and interstitial fibrosis, and IL-6 represents a new factor in the EP4 pathway.

Hydrochloride pioglitazone decreases urinary cytokines excretion in type 2 diabetes

To observe the effects of hydrochloride pioglitazone on urinary cytokine excretion in type 2 diabetes and to explore its possible reno-protective mechanisms. Subjects and Methods. Ninety-eight patients with type 2 diabetes and a fasting blood glucose (FBG) levels between 7.0 and 13.0 mm and glycated haemoglobin A1c (HbA1c) ≥ 7.0% were assigned randomly to receive either the pioglitazone (DP group) or a sulphonylurea (DS group). Another 49 healthy individuals were chosen as normal controls (group NC). At the start of the study and after 12 weeks of treatment, urinary cytokines including monocyte chemoattractant protein-1 (MCP-1), transforming growth factor-β1 (TGF-β1) and vascular endothelial growth factor were measured and were expressed as a ratio of urinary creatinine excretion. Urinary albumin/creatinine ratio, FBG and HbA1c were determined at the same time. The excretion of each urinary cytokine, corrected for urinary creatinine, was significantly increased in both groups of patients with diabetes, compared with normal controls, and after a 12-week treatment were significantly decreased by both therapies but the effect of pioglitazone was statistically greater than with sulphonylureas. Urinary albumin/UCr and both systolic and diastolic blood pressure were decreased significantly by pioglitazone (P < 0.01 or P < 0.05) but not by sulphonylurea treatment (P < 0.05), while there was no significant difference in FBG or HbA1c between two groups. There was a positive correlation between the excretion of cytokines and urinary albumin /UCr (all P < 0.01). This study indicates that pioglitazone reduces urinary albumin excretion by a mechanism that is at least partly independent of blood sugar control. The correlation of urinary albumin excretion with improvement in urinary cytokines suggests that this reno-protective effect of piogliazone in diabetes may be related to local reduction in cytokine activity within the kidney.

Advances in Kawasaki disease

The mainstays of therapy for Kawasaki disease (KD)—IvIG and salicylates—have been employed for years and have proven efficacy. Current advances in KD have mainly been in the areas of understanding pathophysiology and improving diagnosis. The recent recognition of “incomplete KD” is a prime example of the latter. Articles in this issue of The Journal advance our understanding of KD on both fronts. A report from Wu et al in Taiwan reminds us that the vascular inflammation of KD is not limited to the coronary arteries. About 40% of children in this study had evidence of renal inflammation by PET scanning. Of these, there was evidence of increased urinary cytokine excretion and ultrasound evidence of vasculitis in comparison to the children whose PET scans did not show renal inflammation. Although renal problems in KD are generally considered to be self-limited, it will be of great interest to follow such children into adulthood, looking for problems such as hypertension. Another study, from Nomura et al in Japan, looked at the subset of children with KD whose presentation was atypical in being limited to fever and cervical adenopathy. These children tended to be older and had a higher risk of nonresponse to IvIG or the development of coronary artery aneurysms. Although the vast majority of children with fever and cervical adenopathy have bacterial or viral adenitis, we must remember to keep KD in the differential diagnosis. A Severe Form of Kawasaki Disease Presenting with Only Fever and Cervical Lymphadenopathy at AdmissionThe Journal of PediatricsVol. 156Issue 5PreviewTo examine the characteristics of patients with Kawasaki disease (KD) presenting with only fever and cervical lymphadenopathy at admission. Full-Text PDF Urinary Cytokines and Renal Doppler Study in Kawasaki DiseaseThe Journal of PediatricsVol. 156Issue 5PreviewTo investigate whether renal vasculitis is the sole cause or merely a contributing cause of renal inflammation in Kawasaki disease (KD). Full-Text PDF

Oversecretion of Cytokines and Chemokines in Lupus Nephritis Is Regulated by Intraparenchymal Dendritic Cells

Lupus nephritis (LN) occurs in more than one-third of patients with systemic lupus erythematosus. Its pathogenesis is attributed to the glomerular deposition of immune complexes as well as to imbalance of the cytokine homeostasis. In this context, high production of cytokines and chemokines by dendritic cells (DCs) may concur to LN. In addition, urinary cytokine excretion may reflect the accumulation of DCs within glomeruli. DCs are differentiated in both myeloid and plasmacytoid (p) subsets in relation to their typical antigen and chemokine expression. Both subsets migrate in response to chemotactic stimuli because pDCs are susceptible to IL-18 expressed by resident glomerular cells. pDCs bear the IL-18R, and it is conceivable that DCs migrate to the kidney under the attraction of IL-18. Therefore, the depletion of DCs reflects the inflammation severity in LN, whereas measurement of Th1 cytokines may represent an effective tool for monitoring the onset of LN.

Urinary levels of epidermal growth factor, interleukin‐6 and monocyte chemoattractant protein‐1 may act as predictor markers of renal function outcome in immunoglobulin A nephropathy

Urinary cytokine excretion may reflect histological changes in immunoglobulin A nephropathy (IgAN), and their measurement can give information about disease outcome. Thirty-three IgAN patients were prospectively followed for 5.6 +/- 3.1 years. Urinary levels of monocyte chemoattractant protein-1 (MCP-1), interleukin (IL)-6 and epidermal growth factor (EGF) were measured at diagnosis and repeated 1 year later for IL-6 and EGF. Urinary MCP-1 and IL-6 levels were increased significantly, while EGF excretion reduced in IgAN patients, compared to controls. IL-6 urinary levels showed significant positive correlation with chronic histological lesions. Patients were classified into five groups, according to the Haas classification system. MCP-1 and IL-6 urinary levels were increased, whereas EGF levels were reduced in the progression of staging. EGF urinary excretion was a strong predictor factor of disease outcome, significantly correlated with creatinine clearance at time of diagnosis (r = 0.5, P = 0.005), and at the end of follow up (r = 0.6, P = 0.001). Urinary EGF levels measured a year later could predict long-term outcome better, and a cut of 0.05 pg/mg urine creatinine levels could distinguish between progressors and non-progressors. Urinary MCP-1, IL-6 and EGF levels may represent histology in IgAN. EGF excretion can be a predictive marker and its serial measurements may give information about disease outcome and the effect of treatment.

Comparison of Urinary Cytokines after Ingestion of Cranberry Juice Cocktail in Pregnant Subjects: A Pilot Study

Our objective was to evaluate urinary cytokine excretion after daily cranberry or placebo exposure in pregnant women. Four-hour urine samples were collected from 27 pregnant women subjects who were randomized to cranberry juice cocktail or placebo in three treatment arms: A: Cranberry (C) two times daily (C, C; n = 10 pregnant); B: cranberry in the AM, then placebo (P) in the PM (C, P; n = 9 pregnant); and C: placebo two times daily (P, P; n = 8 pregnant). Urinary cytokines were measured using commercially available kits. There was a statistically significant difference in interleukin (IL)-6 of the urinary cytokines between the multiple daily cranberry dosing group (group A [C, C]): median, 3.16 (range, 0.01 to 7.34) and the placebo group (group C [P, P]): 9.32 (0.53 to 29.61 pg/mL; p = 0.038, Kruskal-Wallis test). We concluded that a difference in IL-6 was found in the multiple daily cranberry dosing groups compared with placebo. Lack of differences based on treatment allocation in the other cytokines may be due to beta error. Further studies are planned to evaluate these assays for the assessment of clinical effect.

Mononuclear cells in glomeruli and cytokines in urine reflect the severity of experimental proliferative immune complex glomerulonephritis

Immunohistochemical methods were used to investigate the role of macrophages in the progression of proliferative immune complex glomerulonephritis. The mononuclear cell component of glomerular inflammation was analysed in three different stages of chronic serum sickness, each of which was clearly distinguished by criteria of kidney function. Urinary excretion of the macrophage secretory products interleukin-1 and tumour necrosis factor was also evaluated in relation to the functional severity of kidney disease. T lymphocytes and macrophages began to accumulate in glomeruli at the onset of proteinuria, but not before. Urinary excretion of interleukin-1 also began with proteinuria. Proteinuria increased in direct correlation with increases in the number of glomerular macrophages. Development of the most severe stage of glomerulonephritis, characterized by cachexia, declining kidney function, and necrotizing glomerular pathology, was accompanied by the disappearance of T cells from glomeruli and the expression of highly abnormal phenotypes by most macrophages. In addition, there was a switch from urinary excretion of interleukin-1 to excretion of tumour necrosis factor. The progression of proliferative immune complex glomerulonephritis was associated with qualitative as well as quantitative changes in glomerular macrophage populations. Differentiation and/or activation of those glomerular macrophages may have resulted from local T cell-mediated immunoregulation. Measurements of urinary cytokine excretion provided a reliable means of monitoring disease progression. The local action of tumour necrosis factor probably contributed to declining kidney function in the most severe stage of disease.

Renal Pro-Inflammatory Cytokine Gene Expression in Diabetic Nephropathy: Effect of Angiotensin-Converting Enzyme Inhibition and Pentoxifylline Administration

Background: Recent studies have shown a role for inflammation in the pathogenesis of diabetic nephropathy (DN). Tumor necrosis factor (TNF)-α, interleukin (IL)-1 and IL-6 are cytokines with a prevalent pro-inflammatory activity. Our objective was to study the renal gene expression of TNF-α, IL-1 and IL-6 in DN and their relationship with renal damage assessed by urinary albumin excretion (UAE). In addition, we also investigated the effect of angiotensin-converting enzyme inhibition and pentoxifylline (PTF) administration on these parameters. Methods: After streptozotocin-induced diabetes, rats received either no treatment or therapy with enalapril (EN) or PTF for 8 weeks. Renal expression of pro-inflammatory cytokines was evaluated by real-time polymerase chain reaction. Urinary cytokine excretion and albuminuria were also evaluated. Results: Renal cortical mRNA expression for TNF-α, IL-1 and IL-6 in untreated diabetic rats was 2.4-, 1.2- and 3.4-fold higher than in non-diabetic rats. Kidney weight and UAE were significantly associated with renal mRNA expression of TNF-α and IL-6. Both EN and PTF administration virtually abrogated the overexpression of TNF-α, IL-1 and IL-6, which was associated with a reduction in kidney weight and urinary albumin excretion. Conclusion: The renal expression of the main pro-inflammatory cytokines TNF-α, IL-1 and IL-6 is increased in DN, which is significantly associated with UAE. EN and PTF administration prevented this enhanced expression, leading to a decrease in urinary cytokine excretion and a reduction in albuminuria. These findings provide novel insight into the pathogenic mechanisms of DN, supporting the hypothesis that inflammatory mechanisms play a role in the renal injury secondary to diabetes mellitus.

Application of Antibody Array Technology in the Analysis of Urinary Cytokine Profiles in Patients with Chronic Kidney Disease

Aims: Emerging evidence suggests that the urinary excretion of cytokines is associated with the progression of chronic kidney disease (CKD). However, detection of urinary cytokines in high throughput is still a problem in clinical practice. In this cross-sectional study, we applied a novel proteomic technology, antibody array, to analyze urinary cytokine profiles in patients with CKD. Methods: A total of 10 subjects including 7 CKD patients and 3 normal controls were studied. These patients with CKD were divided into two groups according to the levels of estimated glomerular filtration rate (eGFR): group A (eGFR ≧80 ml/min/1.73 m², n = 3) and group B (eGFR ≤40 ml/min/1.73 m², n = 4). Urine samples taken from age and gender-matched healthy volunteers (n = 3) served as control. Differential excretion of urinary cytokines was determined by human cytokine antibody array (Raybiotech, Norcross, Ga., USA). A 2-fold change in spot intensity compared to controls was considered as significant. In order to check the reliability of the data obtained by antibody array, urinary monocyte chemoattractant protein (MCP) 1 and tumor necrosis factor (TNF) α were determined by using enzyme-linked immunosorbent assay. Results: A total of 15 cytokines varied significantly in urinary samples obtained from patients with CKD compared with normal controls. It was shown that the levels of MCP-1, RANTES, tissue inhibitor of metalloproteinase (TIMP) 1, TNF-α, vascular endothelial growth factor (VEGF), E-selectin, Fas, intercellular adhesion molecule 1, interleukin 2, matrix metalloproteinase (MMP) 2 and transforming growth factor β in patients with CKD at stage 1–2 (group A) were 2- to 5-fold higher than those in normal controls, while the excretion of MCP-1, RANTES, TIMP-1, TNF-α and VEGF was further increased in the patients with renal insufficiency (group B). However, a lower excretion of urinary vascular cell adhesion molecule 1 and platelet-derived growth factor was found in patients with CKD compared with normal controls. Impressively, the urinary MMP-9 excretion was 492-fold higher in group A and 198-fold higher in group B than that in normal controls. The levels of MCP-1 and TNF-α correlated well with the relative n-fold change seen in the antibody array experiments. The rank correlation coefficients (r values) were 0.976 (p < 0.001) and 0.939 (p < 0.001) for MCP-1and TNF-α, respectively. Conclusion: Antibody array could serve as a fast, high-throughput and sensitive tool for detecting the excretion of urinary cytokines. Our study is the first to provide an important information regarding the utility of antibody array in evaluating the role of cytokines in the initiation and progression of CKD.

Tumor necrosis factor-α gene expression in diabetic nephropathy: Relationship with urinary albumin excretion and effect of angiotensin-converting enzyme inhibition

The pathogenic mechanisms and molecular events involved in the development and progression of diabetic nephropathy (DN) are not completely known. Recent data indicate that diabetes includes an inflammatory component that is related to diabetic complications. Tumor necrosis factor (TNF)-alpha, a cytokine with mainly proinflammatory activity, may be synthesized by renal cells. Our objective was to analyze intrarenal TNF-alpha gene expression and its relationship with urinary albumin excretion (UAE). We also investigated the effect of inhibition of angiotensin-converting enzyme on TNF-alpha expression and UAE. Streptozotocin-induced diabetic rats received either no treatment or an angiotensin-converting enzyme inhibitor (enalapril). After eight weeks, renal expression of TNF-alpha was evaluated by real-time polymerase chain reaction. Renal cortical messenger RNA levels of TNF-alpha increased significantly and were twice as high in diabetic rats than in nondiabetic control rats. Enalapril administration nearly completely abolished the increase in TNF-alpha messenger RNA expression to the level observed in control rats. UAE was significantly correlated with urinary levels of TNF-alpha (r=0.68, P<0.05) and with renal TNF-alpha expression (r=0.51, P<0.05). DN was associated with increased renal expression of TNF-alpha and UAE. Enalapril administration prevented this enhanced expression of TNF-alpha and decreased urinary cytokine excretion and albuminuria. These data provide a novel insight into the pathogenic mechanisms of DN, and support the hypothesis that inflammatory mechanisms may play a significant role in the development and progression of renal injury secondary to diabetes mellitus.

Cytokine secretion is impaired in women with diabetes mellitus

As women with diabetes mellitus (DM) have an increased prevalence of asymptomatic bacteriuria (ASB) and it is known that a correlation exists between the increased prevalence of genitourinary tract infection and impaired cytokine production in women infected with Human Immunodeficiency Virus (HIV), we studied urinary cytokine excretion in diabetic women and compared it with that of nondiabetic controls. To evaluate the cytokine secretion capacity of women with DM, both whole blood and isolated monocytes of women with and without DM were stimulated in vitro with lipopolysaccharide (LPS). Lower urinary interleukin-8 (IL-8) and interleukin-6 (IL-6) concentrations (P = 0.1 and P < 0.001, respectively) were found in diabetic women than in nondiabetic controls. A lower urinary leukocyte cell count correlated with lower urinary IL-8 and IL-6 concentrations (P < 0.05). Lower tumour necrosis factor-alpha (TNF-alpha) and IL-6, but comparable interleukin-10 (IL-10) concentrations were found in whole blood (P < 0.04) and isolated monocytes (P = 0.03) of women with DM type 1 compared to women without DM. Diabetic women with ASB have lower urinary IL-6 concentrations than nondiabetic bacteriuric controls. In addition, monocytes of women with DM type 1 secrete lower pro-inflammatory cytokines after stimulation with LPS than monocytes of women without DM. This is not due to an inhibitory effect of the anti-inflammatory cytokine IL-10. This can have important consequences for both host defense, endothelial cell functioning and atherogenesis.