29 JUNE 2005, WEDNESDAY
Smad3-KO mice and SU5416-treated db/db mice. The mesangial matrix expan-sion of diabetes, quantified by electron microscopy, was prevented in Smad3-KO mice but not in SU5416-treated mice. Glomerular VEGF immunostaining, normally increased by diabetes, was unaffected by either Smad3-KO status or SU5416. Finally, nephrin immunofluorescence was diminished in db/db mice and was partially but significantly restored by SU5416.
Thus, overactivity of VEGF signaling, but not necessarily the Smad3 arm of TGF-b signaling, plays a central role in diaTGF-betic alTGF-buminuria. GBM thickening and mesangial matrix expansion can be dissociated from albuminuria, calling into question their pathogenetic roles in albuminuria. Lastly, the reduced nephrin levels of diabetic nephropathy could be provoked by maladaptive VEGF signal-ing.
REVERSAL OF HYDRONEPHROSIS IN THE
MOUSE AS A MODEL FOR RENAL REPAIR
JOHN BERTRAM1, ANITA COCHRANE1, MICHELLE KETT1, WARWICK ANDERSON1, DAVID HUME2, MELISSA LITTLE2, SHARON RICARDO1
Monash University, Australia1, University of Queensland2
The kidney has an inherent ability for recovery and regeneration following acute damage. The present study describes the structural, functional, and microarray analysis of the regenerative potential of the kidney following reversal of ureteral obstruction (R-UUO). Male C57 mice (20–25 g; n = 6/group/time point) under-went unilateral ureteral obstruction (UUO) for 10 days followed by R-UUO for 1, 2, 4, and 6 weeks. Ten days after UUO, mice demonstrated tubular atrophy, medullary ablation, and the development of severe interstitial fibrosis. UUO animals had a 38% fall in total GFR in comparison to sham-operated control animals. Following R-UUO at 2 and 6 weeks, two distinct areas were apparent in the renal parenchyma: one where tubules had repaired and displayed a normal histoarchitecture; and a second where the same kidneys demonstrated tubular and matrix remodeling. Elevated numbers of macrophages were evident in the focal area of remodeling in R-UUO kidneys compared to areas that had under-gone repair (2458.4 ± 393.7/mm2vs. 575.0 ± 202.6/mm2; P < 0.01). However, by 6 weeks there was a progressive decline in total numbers of macrophages in R-UUO kidneys compared to the obstructed kidneys of UUO mice. This was in co-occurrence with reduced type I, III, IV, and V collagen accumulation and resolving interstitial matrix expansion. At this time-point there was a significant but partial restoration of renal function as measured by GFR from individual ureteral catherization of the R-UUO kidneys in comparison to sham-operated control kidneys. In addition, we have used expression profiling to identify genes that are differentially expressed in the remodeling areas of the R-UUO kidneys compared to repaired regions of the same kidneys, in comparison to UUO and control animals. A better understanding of the key events involved in endoge-nous renal repair and remodeling may open the way to new interventions aimed at acceleration of renal regeneration and prevention of scarring.
LONG-TERM OVEREXPRESSION OF
INTERLEUKIN-10 SUPPRESSES MONOCYTE
CHEMOATTRACTANT PROTEIN-1 EXPRESSION
AND INFLAMMATION IN A MODEL OF CHRONIC
WEI MU1, XIAOSEN OUYANG2, DAVID A LONG2, CARLOS A RONCAL J2, ANUPAM AGARWAL3,
OLENA Y GLAUSHAKOVA2,VINCE A. CHIODO2,
WILLIAM W HAUSWIRTH2, BERNARDO RODRIGUEZ-ITURBE4, RICHARD J JOHNSON1,2
University of Florida1, Division of Nephrology, University of Florida, Gainesville,
USA2; Division of Nephrology, University of Alabama, Birmingham, USA3;
Division of Nephrology, University of Maracaibo, Maracaibo, Venezuela4
Interleukin-10 (IL-10) is a cytokine that plays a pivotal role in the regulation of T-cell immune responses. Previous studies have shown that IL-10 may diminish
Experimental Chronic Kidney Disease
CASPASE INHIBITION REDUCES TUBULAR
APOPTOSIS AND PROLIFERATION AND SLOWS
DISEASE PROGRESSION IN POLYCYSTIC KIDNEY
YUNXIA TAO1, JUN KIM, ROBERT W. SCHRIER AND
CHARLES L. EDELSTEIN1
University of Colorado Health Sciences Center1
Increased apoptosis is a characteristic feature of polycystic kidney disease (PKD). The aim of the present study was to determine the effect of caspase inhibition on tubular cell apoptosis and proliferation, cyst formation and renal failure in the Han: SPRD rat model of PKD. Heterozygous (Cy/+) and littermate control (+/+) male rats at 3 weeks of age were treated with the caspase inhibitor, IDN8050 (10 mg/kg/d) via a minipump or vehicle for 5 weeks. On immunoblot analysis the active form of caspase-3 in kidney was significantly reduced in rats treated with IDN8050. The number of TUNEL positive tubular cells per cyst in the cortex was 0.3 in vehicle-treated Cy/+ and 0.01 in IDN8050 treated Cy/+ (P < 0.05). The 2 kidney/total body weight ratio was 0.9 in vehicle-treated +/+ (n = 8), 2.1 in vehicle-treated Cy/+ (p < 0.001 vs +/+) and 1.6 in IDN8050-treated Cy/+ (P < 0.001 vs Cy/+ n = 6). Cyst volume density (CVD)(%) was 0.4 in vehicle-treated +/+ (n = 4), 43 in vehicle-treated Cy/+ (p < 0.001 vs +/+) and 30 in IDN8050-treated Cy/+ (P < 0.001 vs Cy/+ n = 6). BUN was 23.5 in vehicle-treated +/+ (n = 4), 38 in vehicle-vehicle-treated Cy/+ (p < 0.001 vs +/+) and 26 in IDN8050-treated Cy/+ (P < 0.001 vs Cy/+ n = 4). The number of PCNA posi-tive cells per non-cystic tubule in the cortex were 0.04 in vehicle-treated +/+, 0.8 in vehicle-treated Cy/+ (p < 0.001 vs +/+) and 0.15 in IDN8050-treated Cy/+ (P < 0.05 vs Cy/+ n = 4), and the number of PCNA positive cells per cyst in the cortex was 1.0 in vehicle-treated Cy/+ rats and 0.12 in IDN8050 treated Cy/+ (P < 0.05, n = 4). In summary, in a rat model of PKD, caspase-inhibition with IDN-8050 1) decreases apoptosis and proliferation in cystic and non-cystic tubules; 2) inhibits renal enlargement by 44% and cyst volume density by 29%; and 3) atten-uates the loss of kidney function.
VEGF SIGNALING, BUT NOT SMAD3 SIGNALING,
ENGENDERS ALBUMINURIA IN DIABETIC
NEPHROPATHY, POSSIBLY VIA ALTERATIONS IN
SHELDON CHEN1, SUN HEE SUNG1, NICHOLAS J. LAPING2, FUAD N. ZIYADEH1
University of Pennsylvania Renal Division Philadelphia United States1, Urogenital
Biology, CVU CEDD, GlaxoSmithKline, King of Prussia, PA, USA2
The pathogenesis of albuminuria in diabetic nephropathy remains mysterious. Our earlier work did not demonstrate improvement of albuminuria by neutraliz-ing TGF-b in type 2 diabetic db/db mice. Subsequently, neutralization of VEGF has successfully ameliorated albuminuria in rodent models of type 1 and type 2 diabetes. To explore the roles of TGF-b and VEGF without resorting to an antibody-based approach, Smad3-knockout mice were made diabetic with strep-tozotocin, and a pan-VEGF receptor inhibitor, SU5416, was administered to db/db mice.
Smad3-KO mice and their age-matched wildtype controls were randomized to receive vehicle or streptozotocin and remained diabetic for six weeks. Eight-week-old db/db and control db/m mice were injected intraperitoneally with vehicle or 2 mg/kg SU5416 twice-a-week for 8 weeks. In both experimental groups, the urine albumin excretion (UAE), measured by ELISA, increased with diabetes, rising ~3-fold in the STZ-wildtype mice and ~4-fold in the db/db mice. The increased UAE of diabetes was not improved in the diabetic Smad3-KO mice, but was significantly ameliorated in the db/db mice by SU5416 treatment. Based on structure-function studies, histologic parameters that correlate with albuminuria were measured. Diabetic GBM thickening was prevented in both
EX VIVO PHENOTYPIC SWITCH DETERMINES
DAMAGING VERSUS PROTECTIVE EFFECT OF
MACROPHAGE IN MURINE
YING WANG1, YIPING WANG1, GUOPING ZHENG2, YUET-CHING TAY, XIAOHONG QIN, DEEPIKA MAHAJAN, STEPHEN I ALEXANDER, DAVID C.H. HARRIS
Centre for Transplantation and Renal Research, The University of Sydney at Westmead Millenium Institute1; Centre for Kidney Research Children’s Hospital at
Westmead NSW 2145 Australia.2
Background: Macrophage infiltration is a constant feature of glomerular and
tubulointerstitium in both human and experimental kidney disease. We previ-ously showed that depletion of macrophages partially reduced renal structural and functional injury in murine adriamycin nephropathy (AN). Here we investigated whether macrophage phenotype was important in determining its effect on kidney disease in the absence of T and B cells.
Methods: AN was produced in male SCID mice (deficient in T and B cells) by
adriamycin (ADR 5.2 mg/kg single/iv). Macrophages separated from normal female Balb/C mice were incubated with or without LPS (2.5 mg/ml for 2 hours, Type 1 MF) or with IL-4 and IL-13 (10 ng/ml each for 48 hours, Type 2 mF). One million cells were transferred into treated mice by a single iv at day 5 after ADR. Seven mice in each of groups A (ADR + saline treated), B (ADR + unstimulated MF), C (ADR + Type 1 MF) and D (ADR + Type 2 MF) were sacrificed and renal function and histopathological features were assessed on day 28.
Results: Type 1 MF worsened, Type 2 MF ameliorated and unstimulated MF
did not affect renal function and histology compared with the mice on adriamycin alone.
ADR Unstimulated Type 1 Type 2
MF MF MF Creatinine Clearance 22.5 ± 6.3 22.6 ± 7.2 12.3 ± 5.3 48.3 ± 6.8 (mL/min) Glomerular Sclerosis 1.7 ± 0.5 1.7 ± 0.4 2.6 ± 0.5 0.7 ± 0.3 (0–3) Interstitial Expansion 1.5 ± 0.6 1.6 ± 0.4 2.6 ± 0.7 0.8 ± 0.4 (0–3) Tubular Atrophy (0–3) 1.5 ± 0.6 1.6 ± 0.4 2.6 ± 0.4 1.0 ± 0.3 Overall Injury (0–3) 1.6 ± 0.7 1.7 ± 0.4 2.5 ± 0.5 1.2 ± 0.6
For all comparisons among unstimuated, Type 1 MF and Type 2 MF groups (ANOVA), P < 0.01.
Conclusions: Reconstitution of Type 1 MF but not unstimulated macrophages
can substantially augment renal injury in AN, whereas, reconstitution with Type 2 MF reduces injury in AN. These results demonstrate that ex vivo modulation of macrophage phenotype can directly affect the capacity of these cells to modify renal injury.
Epidemiology and Pathogenesis of Chronic
PREDOMINANT EFFECTS OF KIDNEY DISEASE
(KD) ON EXCESS MORTALITY IN PIMA INDIANS
WITH OR WITHOUT TYPE 2 DIABETES
MEDA E. PAVKOV1, PETER H. BENNETT1, MAURICE L. SIEVERS1, JONATHAN KRAKOFF1, DESMOND E. WILLIAMS2,
WILLIAM C. KNOWLER1, ROBERT G. NELSON1, NIDDK1, CDC2
We examined the effects of KD on mortality from natural causes in non-diabetic and diabetic Pima Indians aged ≥45 years, between 1965–2001.
Deaths per person-years (pyrs) of follow-up were stratified in a time-dependent manner into categories of 1) no proteinuria and normal serum creatinine (SCr), 2) proteinuria (≥0.5 g/g) and normal SCr, 3) high SCr (SCr ≥ 133 mmol/L (1.5 mg/dl) in men, ≥124 mmol/L (1.4 mg/dl) in women), or 4) renal replacement therapy (RRT).
inflammatory and proliferative responses in animal models of glomerulonephri-tis. However, to date there have been no studies on the potential benefits of IL-10 in chronic renal disease. Therefore, long-term expression of IL-IL-10 in rats was induced by administration of an adeno-associated virus IL-10 (AAV-IL-10) vector. Control rats received a similar dose of AAV-GFP. 4 weeks after injection, IL-10 levels (measured by ELISA) were significantly elevated (346 ± 64 versus 64 ± 6 pg/ml, IL-10 vs controls, n = 6 per group, p = 0.001). At this time rats underwent 5/6 nephrectomy, 8 weeks after of surgery, the rate were sacrificed for RNA, protein and immunohistochemical analysis. At sacrifice, IL-10 levels remained 6-fold higher than controls (p < 0.01). IL-10 treated animals had improved renal function compared to the control-GFP group with significantly less proteinuria (p < 0.05), serum creatinine (p < 0.05) and an improved creati-nine clearance rate (p < 0.01). Renal interstitial infiltration was significantly inhibited by IL-10 administration as assessed by numbers of CD4+, CD8+, ED-1 and OX-62 positive cells (approximately 40–60% reduction, p < 0.05). IL-ED-10 administration also decreased renal IFN-g and IL-2 mRNA (by quantitative real-time PCR) indicating that IL-10 is primarily involved in suppressing the Th1 response in chronic renal disease. Finally, to assess the potential mechanism of reduced inflammation in the IL-10 treated animals, we measured levels of a chemokine, monocyte chemoattractant protein-1 (MCP-1), a potent chemoat-tractant for macrophages and T-cells. Kidney MCP-1 mRNA and protein levels were suppressed by IL-10 treatment as measured quantitatively by real-time PCR and ELISA respectively (p < 0.05). Collectively, these results indicate that IL-10 inhibits the renal interstitial inflammation in chronic renal disease potentially through lowering MCP-1 expression in kidney leading to an improved renal function.
TRANSGLUTANIMANSE (TG) INHIBITION
AMELIORATES THE DEVELOPMENT OF
EXPERIMENTAL DIABETIC NEPHROPATHY (DN)LINGHONG HUANG1, JOHN HAYLOR1, MARIE FISHER1, ZOE HAU1, MARTIN GRIFFIN2, MEGUID EL NAHAS1, TIM JOHNSON1, MARTIN GRIFFIN2
Sheffield Kidney Institute1, Department of Life Sciences, Nottingham Trent
In kidney disease increased extracellular Tg leads to e(g-glutamyl)-lysine crosslinking within the Extracellular Matrix (ECM), altering ECM homeostasis and causing expansion of the basement membranes & scarring. In the acute 5/6th subtotal nephrectomy model of renal scarring, Tg inhibitors maintained renal function by prevention of tissue scarring. Here we demonstrate long term anti-Tg therapy in a chronic rat model of DN.
Following uninephrectomy (UNx) to accelerate DN, 50 mmol/L Tg inhibitor NTU281 was infused continually into the left kidney from an osmotic minipump. Diabetes was induced 7 days later using 35 mg/kg of Streptozotocin (STZ). 4 experiment groups of normal, UNx, untreated diabetics (DM) and DM treated with NTU281 were used. Blood glucose was maintained between 20–25 mmol/L with insulin implants. Blood, urine and tissue samples were collected 1, 4 and 8-months post STZ. Kidney function was assessed by creatinine clearance and renal scarring by image analysis of Masson’s Trichrome stained sections. Active TGFb was measured using a PAI-1 reporter bioassay and specific ECM proteins by immunofluorescence.
Creatinine clearance decreased by 48.6% in DM rats by 8-months. In the NTU281 treated group, it remained similar to the non-diabetic UNx group. An increase in proteinuria was detected in DM rats; however, proteinuria was reduced by >50% in the treated groups at 4 and 8-months. Histological examination of kidneys 8-months post STZ indicated extensive glomerulosclerosis (1.16 ± 0.58) and tubulointerstitial (1.3 ± 0.86) scarring in untreated diabetics. In comparison diabetic animals treated for 8 months with NTU281 had reduced scarring in both the glomeruli (0.25 ± 0.10) and tubulointerstitium (0.29 ± 0.13) resulting from lower deposits of Collagens I, III and IV. This was associated with lower active TGFb levels.
Tg inhibition from the onset of diabetes in experimental DN improves kidney function and reduces renal scarring over an 8-month period. This type of therapy offers potential for translation to the clinical environment.
Conclusion: Death rates increased with worsening kidney function in both
non-diabetic and non-diabetic subjects and were similar in nonnon-diabetic and non-diabetic sub-jects without KD. KD was associated with excess mortality from DN, CVD, and infections in diabetic, and from infections in nondiabetic subjects.
THE PROGNOSIS OF CHRONIC KIDNEY DISEASE
K/DOQI STAGE 3: A TEN YEAR
POPULATION-BASED STUDY OF THE EFFECTS OF GENDER
BJØRN ODVAR ERIKSEN1, OLE CHR. INGEBRETSEN2
Department of Nephrology, University Hospital of North Norway1; Department of
Clinical Chemistry, University Hospital of North Norway, Tromsø, Norway2
The increasing demand for renal replacement therapy makes it important to investigate the prognoses of the earlier stages of chronic kidney disease (CKD). We examined change in glomerular filtration rate (GFR), and patient and renal survival in CKD K/DOQI stage 3 in Tromsø, a well-defined European commu-nity with a population of 58,000.
All patients with estimated GFR between 30 and 59 ml/min/1.73 m2 for more than three months during a ten year study period were identified from a plete database of all 248,560 measurements of serum creatinine made in the com-munity in the study period. Change in GFR was estimated for each patient using a linear model. A complete follow-up of patient and renal survival was obtained from hospital databases.
3047 patients were included. The mean number of measurements of creatinine for each patient was 14; the mean observation time 50 months. Mean change in GFR was -1.02 ml/min/1.73 m2/year (SD 8.22). 61% experienced a decline in GFR. Ten year cumulative incidence of renal failure was 0.04 (95% CI 0.03 to 0.06); mortality 0.51 (95% CI 0.48 to 0.55). Slower decline in GFR and better patient and renal survival were seen in women. Increasing age was associated with faster decline, higher mortality and a lower cumulative incidence of renal failure because it was pre-empted by death.
Our population-based estimate of GFR decline was markedly slower than in pre-viously studied selected CKD patients. Different prognoses in identifiable subgroups make it difficult to establish guidelines with a uniform approach to
all patients. Risk factors for predicting which patients have progressive disease should be included in the definitions of stages, as i.e. proteinuria. Although included in the definition of K/DOQI stages 1 and 2, definitions of the advanced stages have been made without taking proteinuria into account. This needs to be reconsidered.
RACE DIFFERENCES IN THE DISTRIBUTION OF
NOVEL CARDIOVASCULAR RISK FACTORS
AMONG PERSONS WITH CHRONIC KIDNEY
DISEASE: FINDINGS FROM THE THIRD
NATIONAL HEALTH AND NUTRITION
HOANG THANH NGUYEN1, MURTHY V.R. BHAMIDIPATI1, AUSTIN G. STACK2
Department of Internal Medicine, Division of Renal Diseases and Hypertension, University of Texas at Houston Medical School, U.S.A.1; Regional Kidney Center,
Letterkenny and Sligo General Hospitals, North Western Health Board, Ireland2
The prevalence of clinical coronary disease differs by race among persons with chronic kidney disease (CKD). Whether these can be accounted for by differ-ences in the distribution of novel cardiovascular risk factors is unclear. We compared the prevalence of novel inflammatory [C-reactive protein (CRP), white blood cell count (WBC)] and prothrombotic [apolipoprotein A1 (Apo A1), and apolipoprotein B (Apo B), homocysteine (HCY), lipoprotein (a) (Lp (a)), and plasma fibrinogen] risk factors among Whites, Blacks and Hispanics with reduced kidney function in the Third National Health and Nutrition Exam-ination Survey, a population-based survey of the non-institutionalized U.S. population. CKD was defined as a glomerular filtration rate <60 ml/min per 1.73 m2from the 4-variable Modification of Diet in Renal Disease Study equa-tion. Pathological levels for each factor were defined as those within the highest quartile with the exception of ApoA1 (lowest quartile) and CRP (≥10.0 mg/L). Age-standardized prevalence for each factor was computed and compared by race while weighted logistic regression was used to compare the odds-ratios.
Age-Standardized Prevalence of Pathological levels of Novel Cardiovascular Risk-Factors by GFR and Race
Risk-Factors N >60 ml/min CKD (<60 ml/min)
p-White Black Hispanic White Black Hispanic value*
CRP 15,200 6.8 12.6 8.4 17.3 18.8 10.4 NS (>10 mg/L) WBC 15,033 25.4 18.1 29.4 41.9 17.3 49.5 <.005 (>75th) Fibrinogen 8,844 22.6 31.2 24.7 39.0 61.1 73.2 <.005 (>75th) Apo A1 7,343 24.4 16.8 27.3 45.5 27.1 24.2 NS (<25th) Apo B 7,360 36.6 22.5 29.4 61.2 31.8 44.7 <.05 (>75th) Lp(a) 7,769 20.9 57.8 15.6 22.6 60.6 40.8 NS (>75th) HCY 6,790 24.8 25.5 19.7 77.9 49.2 95.3 <.0001 (>75th)
*p-value is from t-text of polynominal linear or quadratic contrast across race categories for the CKD population NS = Not statistically significant.
Subjects with CKD had higher prevalence of abnormal levels of almost all risk factors than subjects with GFR >60. Pathological elevations in WBC, fibrinogen and HCY were greatest in Hispanics compared with other races. Conversely, pathological levels of CRP and ApoA1 were lowest in Hispanics compared with other races. Blacks had the lowest prevalence of pathological levels of Apo B. Compared to Whites, Blacks were 1.8 and 3.7 times more likely to have abnor-mal levels of fibrinogen and Lp (a) but 38% less likely to have abnorabnor-mal level of WBC, while Hispanics were 1.7 and 2.6 times more likely to have abnormal levels of Apo A1 and fibrinogen in the CKD population. Whether these translate into measurable differences in cardiovascular event rates needs further study.
Fig. 1 Death rates from natural causes, stratified by diabetes/duration in four
cat-egories of kidney function. There was a strong effect of kidney function in all categories of diabetes duration, but lesser effects of duration for any category of kidney function.* = insufficient data
Among 1,990 subjects 55.8% had type 2 diabetes at baseline. In nondiabetic sub-jects age-sex-adjusted death rates increased from 23.8/1,000 pyrs (95% CI 20.8–26.8) in those without KD to 84.5/1,000 pyrs (95% CI 1.6–167.4) in sub-jects with high SCr (p < 0.0001), with excess mortality primarily from infections. Among diabetic subjects, death rates rose from 25.3/1,000 pyrs (95% CI 22.3–28.2) in subjects without KD, to 209/1,000 pyrs (95% CI 171.1–246.3) in those with RRT (p < 0.0001). Death rates were associated with KD regardless of diabetes duration (Figure 1). High SCr was associated with higher death rates from cardiovascular disease (CVD), diabetic nephropathy (DN), and infections.
IMPACT OF DIABETIC NEPHROPATHY AND
ANGIOTENSIN II RECEPTOR BLOCKADE ON
URINARY POLY-PEPTIDE PATTERNS
KASPER ROSSING1, HARALD MISCHAK2, HANS-HENRIK PARVING3, PER K. CHRISTENSEN1, MICHAEL WALDEN4, MEIKE HILLMANN4, THORSTEN KAISER4
Steno Diabetes Center1, Mosaiques diagnostics & therapeutics AG, Hannover,
Germany2, Steno Diabetes Center, Niels Steensen Vej 2, Gentofte, Denmark3,
Mosaiques diagnostics & therapeutics4
Background: New insights into the pathogenesis and treatment of diabetic renal
disease may emerge from recent advances in proteomics using high-throughput mass spectrometry (MS) of urine.
Methods: Using a combination of online capillary electrophoresis(CE) and MS
we evaluated urinary polypeptide patterns(UPP) in 4 groups of type 2 diabetic patients matched for age, gender and diabetes duration including; 20 normoal-buminuric patients with and 20 without diabetic retinopathy(DR), 20 microal-buminuric patients with DR and 18 macroalmicroal-buminuric patients with DR. Furthermore, changes in UPP during treatment with the angiotensin II receptor blocker candesartan were evaluated in the macroalbuminuric patients in a ran-domized double-blind, cross-over trial where each patient received treatment with placebo, candesartan 8, 16 and 32 mg daily each for two months.
Results: Overall 4551 different polypeptides were found in the samples. UPPs
were comparable in normo- (with and without DR) and microalbuminuric patients whereas distinct differences were found in macroalbuminuric patients. Differences in UPP between normo- and macroalbuminuric patients permitted the establishment of a ‘diabetic renal damage’ (DRD) pattern consisting of 113 polypeptides. Eleven of these polypeptides had been sequenced and identified. Candesartan treatment in macroalbuminuric patients significantly changed 15 of the 113 polypeptides in the DRD pattern towards levels in normoalbuminuric patients. Change in the DRD pattern was not candesartan dose-dependent but individual changes correlated with changes in urinary albumin excretion at each dose level.
Conclusions: CE-MS serves as a fast and sensitive tool for identification of
bio-markers and urinary polypeptide patterns specific for macroalbuminuric type 2 diabetic patients and may be used to explore and monitor renoprotective effects of angiotensin II receptor blockade.
UP-REGULATION IN THE KIDNEY AND GENETIC
POLYMORPHISM OF MUC20, A REGULATOR OF
MET SIGNALING CASCADE, IN PATIENTS WITH
ICHIEI NARITA1, BASSAM ALCHI2, FUMINORI SATO3, DAISUKE KONDO, ASA OGAWA, YUTAKA TSUBATA,
MINORU SAKATSUME, TADASHI YAMAMOTO, FUMITAKE GEJYO Division Of Clinical Nephrology And Rheumatology, Niigata University, Japan1;
Division of Clinical Nephrology and Rheumatology and Institute of Nephrology2,
Niigata University Graduate School of Medical and Dental Sciences3
MUC20, mucin protein 20, was isolated as a gene up-regulated in the renal tissue of IgA nephropathy (IgAN). The functional analyses of MUC20 have demon-strated that it is a novel negative regulator of the HGF-induced Grb2-Ras pathway. It has been suggested that oligomerization of MUC20 caused by either an overproduction or an unknown factor(s) leads to the association with Met. In human MUC20, the repeat numbers of the extracellular tandem domain, which may have an influence on the oligomerization, showed a divergence, with 2 to 6-repeat types in several human cell lines. To clarify the role of MUC20 in human kidney diseases, we analyzed the expression of MUC20 in the kidney tissues of primary glomerulonephritis by in situ hybridization and investigated a possible association of the tandem repeat polymorphism of MUC20 with renal survival in 236 patients with histologically- proven IgAN. In normal kidneys, MUC20 expression was localized only in distal tubules. In addition, glomerular podocytes and Bowman’s capsule were positive for MUC20. In contrast, it was expressed in proximal tubules in addition to distal tubules in renal tissues of IgAN, whereas change in glomerular expression was not significant. The prognosis of IgAN patient with 5 or 6 of tandem repeat of MUC20 was significantly better than those without (P = 0.001). The tandem repeat polymorphism of MUC20 was an independent risk factor for progression even after adjusting with other clinical risk factors, including hypertension, urinary protein excretion. MUC20 regulates the Met signaling cascade, which is implicated in tubular repair and
ASYMMETRIC DIMETHYLARGININE (ADMA) AND
PROGRESSION OF CHRONIC KIDNEY DISEASE:
THE MILD TO MODERATE KIDNEY FAILURE
JAN T. KIELSTEIN1, FLORIAN KRONENBERG2, DANILO FLISER1, STEFANIE M BODE-BOEGER3, EBERHARDT RITZ4
Department of Nephrology, Medical School Hannover, Germany1; Institute Med.
Biol. & Hum. Gen., University of Innsbruck, Innsbruck, Austria2; Institute of
Clinical Pharmacology, University Hospital Ott-von-Guericke University, Magdeburg, Germany3; Department of Internal Medicine, Ruperto-Carola
University, Heidelberg, Germnay4
Background: Increased blood levels of the endogenous NOS inhibitor
asym-metric dimethylarginine (ADMA) have been proposed to contribute to the pro-gression of chronic kidney disease (CKD). We assessed the influence of ADMA and other putative progression factors in a prospective multi-centre study in 227 Caucasoid patients with non-diabetic kidney disease.
Study population and Methods: Patients were recruited from eight nephrology
departments in Germany, Austria, and South Tirol. Major exclusion criteria were diabetes mellitus, immunosuppressive treatment, serum creatinine above 6 mg/dl, nephrotic syndrome and malignancy. ADMA concentrations were measured with liquid chromatography-mass spectrometry method. In addition, glomerular filtration rate (GFR) was assessed using the iod-thalamate clearance technique. After baseline examination patients were followed prospectively until doubling of serum creatinine, terminal renal failure necessitating renal replacement therapy, or end of the 82 months observation period.
Results: At baseline plasma ADMA levels were significantly correlated with age
(r = 0.281), serum creatinine (r = 0.595), GFR (r = -0.591), proteinuria (r = 0.184), hemoglobin (r = -0.336) and PTH (r = 0.586) (all p < 0.01). A total of 177 patients (78%) in the baseline cohort could be assessed during the follow-up period. The median follow-follow-up time was 54 [1–82] months, and during this period 65 patients reached a progression endpoint. We performed Cox regression with variables which were significantly different in patients who progressed during follow-up only baseline serum creatinine (OR = 30.36; 95% CI 13.09–70.34; p < 0.001) and ADMA (OR = 6.35; 95% CI 2.94–13.70; p < 0.006) were significantly associated with progression. In patients with supra-median ADMA values progression was significantly (p < 0.0001) faster, and their mean follow-up time to a progression endpoint was to 54.6 (95% CI 48.9–60.4) months as compared to 71.3 (95% CI 65.7–76.9) months in patients with infra-median ADMA levels (Figure).
Conclusion: The results of this prospective study in a sizable cohort of patients
with non-diabetic kidney disease point to ADMA as a novel risk factor in the progression of renal disease.
tion under pathological condition in glomerulonephritis. Factors that regulate the function of MUC20 may be useful therapeutic agent for progression of renal injury.
OF PPARg BLOCKS
THIAZOLIDINEDIONE-INDUCED FLUID RETENTION
TIANXIN YANG1, AIHUA ZHANG1, RAOUL D. NELSON1, FRANK J. GONZALEZ2, DONALD E. KOHAN1, HUI ZHANG1 University of Utah1, National Institute of Health2
The peroxisome proliferator-activated receptor subtype g (PPARg) ligands, namely the synthetic insulin-sensitizing thiazolidinedione compounds (TZDs), have demonstrated great potential in the treatment of type II diabetes. However, their clinical applicability is limited by a common and serious side effect of edema. To address the potential role of the distal nephron in TZD-induced fluid retention, the present study generated mice with collecting duct (CD)-specific deletion of PPARg gene (termed PPARg KO) by genetic cross between PPARg floxed (PPARgf/f) and AQP2-CreTag mice. A 7-day rosiglitazone (RGZ) treat-ment consistently increased body weight in PPARgf/fmice but not in PPARg KO mice. Following the RGZ treatment, PPARgf/fmice exhibited severe plasma volume expansion as reflected by significant decreases in hematocrit and plasma aldosterone levels, and significant increases in plasma volume measured by Evans blue technique. In contrast, the RGZ-induced plasma volume expansion was remarkably blunted in PPARg KO mice. The volume expansion in RGZ-treated PPARgf/f mice was preceded by a positive sodium balance, whereas PPARg KO mice maintained normal sodium balance throughout the entire experimental period. RGZ significantly stimulated sodium transport as reflected by decreases in the transepithelial resistance in the primary culture of CD cell-derived PPARgf/f mice, while the stimulation was completely blocked in PPARg KO cells. We conclude that TZD-induced fluid retention is mediated by activation of PPARg in the CD.
REGULATION OF RENIN SECRETION IN MICE
WITH CRE-RECOMBINASE-MEDIATED DELETION
OF JUXTAGLOMERULAR GSALPHA
SOO MI KIM1, LIMENG CHEN1, YUNING HUANG, MIN CHEN1, MARIA LUISA SEQUEIRA LOPEZ2, R. ARIEL GOMEZ2, S. LEE WEINSTEIN1, JOSIE P. BRIGGS1, JURGEN SCHNERMANN1 NIDDK, NIH1, University of Virginia2
The present experiments were performed to assess the role of Gsa as a manda-tory transducer of G protein-coupled receptor activation in the regulation of renin secretion. Since a complete Gsa knockout is embryonic lethal, we gener-ated mice in which Gsa was elimingener-ated selectively in renin-expressing cells by cell-specific cre-mediated recombination. These mice were the offspring of a cross between mice heterozygous for a knockin of cre recombinase into the Ren1d locus placing cre under the control of the renin promoter (Sequeira Lopez et al., Dev. Cell 6:719, 2004) and mice in which exon 1 of the Gsa gene was flanked by LoxP sites (Chen a.Weinstein, NIDDK, unpublished). Ren1d-Cre/GsF.F mice were viable and showed no obvious anatomical abnormalities. Cre-mediated DNA recombination and inactivation of Gsa was found in the kidney cortex as well as in the renal medulla. Basal plasma renin concentration (PRC; ng AngI/ml/hr) was extremely low, and it did not significantly change with the administration of furosemide (43 ± 28 vs. 81 ± 94), isoproterenol (76 ± 41 vs. 387 ± 129), or hydralazine (172 ± 54 vs. 260 ± 296). In control Ren1d/GsF.F mice furosemide increased PRC from 926 ± 211 to 5652 ± 1888, isoproterenol from 1041 ± 257 to 1556 ± 384, and hydralazine from 1007 ± 258 to 1672 ± 416 (p > .05 for all inter-ventions). In agreement with medullary Gsa inactivation Ren1d-cre/GsF.F mice had an AVP-resistant concentrating defect. We conclude that ambient as well as macula densa- and baroreceptor-dependent renin release is dominated by Gs-coupled receptor signaling. Thus, cyclic AMP appears to be the major intracel-lular messenger regulating renin synthesis and release.
MACULA DENSA REGULATION OF RENIN
SECRETION AND PREGLOMERULAR RESISTANCE
IN MICE LACKING THE B-ISOFORM OF THE
NA/K/2CL COTRANSPORTER NKCC2
HAYO CASTROP1, MONA OPPERMANN1, YUNING HUANG1,
DIANE MIZEL1, CHUXIA DENG1, JOSIE P. BRIGGS1,
JURGEN SCHNERMANN1 NIDDK, NIH1
NaCl transport by NKCC2 in the macula densa (MD) is an early step in the mechanism by which changes in luminal NaCl concentration are translated into changes of renin secretion and preglomerular resistance. To further address these questions we generated mice lacking the B-isoform of NKCC2 believed to be mainly expressed in the MD. Genomic DNA analysis showed predicted muta-tions of NKCC2B-specific sequences in NKCC2B-/- mice. Sequencing of tran-scripts revealed correctly spliced NKCC2A and NKCC2F mRNA at normal expression sites (NKCC2A: OM outer stripe = OM inner stripe > cortex; NKCC2F: OM inner stripe >> OM outer stripe >>>> cortex). Expression of NKCC2F was reduced in NKCC2B-/- mice. Two mutated NKCC2B transcripts were found in NKCC2B-/- mice predicted to result in a truncated and non-functional NKCC2B protein. NKCC2B-/- mice were viable and showed no morphological abnormalities. Ambient urine osmolarity was lower in NKCC2B-/- than in wild type controls (1243 ± 128 vs.1844 ± 149 mosm/l; n = 21 and 15; p = .03 =). Basal plasma renin concentration (PRC; ng angiotensin I/ml/hr) was reduced in NKCC2B-/- (1040 ± 135 vs. 1488 ± 136; n = 12 and 16; p = .03), while increases of PRC with a low salt diet (0.003% NaCl) and decreases of PRC with a high salt diet (8% NaCl) were maintained. In loops of Henle perfused in vivo with 6 nl/min, distal Cl concentration was higher (70 ± 4 vs. 43 ± 3.5 mEq/l; p < .0001) and Cl absorption was lower in NKCC2B-/- than wild type mice (653 ± 17 vs. 747 ± 12 pEq/min; p = .00015). Maximum tubuloglomerular feedback responses were not different between NKCC2B-/- and wild type mice, but TGF curves were right-shifted. We conclude that absence of NKCC2B reduces the diluting capacity of the TAL, reduces net Cl absorption along the loop of Henle, and desensitizes the TGF response.
N-TERMINAL SORTING SIGNAL MEDIATES
POLARIZED TRAFFICKING OF AQUAPORIN 3
(AQP3) WATER CHANNEL
TATEMITSU RAI1, SEI SASAKI1, SHINICHI UCHIDA1 Tokyo Medical and Dental University1
Epithelial renal collecting duct cells express multiple types of aquaporin (AQP) water channels in a polarized fashion. AQP2 is specifically targeted to the apical cell domain, whereas AQP3 and AQP4 are expressed on the basolateral mem-brane. Although it is crucial that different types of AQPs are sorted to their proper polarized membrane domains for efficient cell function, little is known about the molecular mechanisms that govern the membrane targeting of AQPs. In the present study, we examined the polarized trafficking and surface expres-sion of AQP3 in MDCK cells to identify regions that specify polarized targeting. Human AQP3 cDNA was subcloned into pcDNA3.1 expression vector. (Invitrogen) and mutations were introduced by Quikchange Mutagenesis kit (Stratagene). When expressed in MDCK cells, wild type AQP3 was targeted to the basolateral membrane resembling its proper localization in vivo. A potential sorting signal consisting of tyrosine and dileucine based motifs was identified in the N-terminal of AQP3. When mutations were introduced in the sorting signal, basolateral targeting of mutant AQP3s was interfered and mutant AQP3s remained in the cytoplasm. Next, AQP2-AQP3 chimeras were generated by Seamless cloning kit (Stratagene) in which the entire N-terminal of AQP2 was substitute to that of AQP3. AQP2 with the N-terminal of AQP3 was consitu-tively mis-localized in the basolateral membrane, whereas mutations in the N-terminal signal abolished the effect. From these results, we conclude that the N-terminal sorting signal mediates basolateral targeting of AQP3.
THEME 1: FUNDAMENTALS OF
EVALUATION OF THE HEMODYNAMIC
PARAMETERS DURING NORMAL PREGNANCY IN
RATS: ROLE OF RENAL WATER AND
MIRIAN A BOIM1, NAYDA P ABREU1, WELLINGTON QUINTINO1, JOISSE C.B. TARDIN1, RUY R. CAMPOS1, CASSIA T. BERGAMASCHI1, NESTOR SCHOR1
Universidade Federal de São Paulo1
Water and salt retention is a typical condition during normal pregnancy. The kidneys are involved in the extracellular volume (ECV) expansion. Thus the aim of the present study was to evaluate if the alterations in ECV observed during normal pregnancy is paralleled with changes in the mRNA expression levels of renal electrolytes and water transporters. Pregnancy was induced in normal Wistar rats and was detected by the presence of vaginal sperm (group P, n = 6). Age matched virgin rats served as control (C, n = 6). The hemodynamic para-meters including blood pressure (BP), heart rate (HR), stroke volume (SV), total peripheral resistence (TPR) and cardiac output (CO) were analyzed at 14thday of pregnancy. The CO was estimated by the termodilution method. The mRNA expression levels of Na/H exchanger (NHE3), Na/K/2Cl cotransporter (BSC) and the aquaporin 2 (AQP2) were analyzed in the kidney tissue by RT-PCR tech-nique. Results were expressed as densitometric units using b actin mRNA as the endogenous control. CO increased in P group compared with C group (107 ± 7 vs 84 ± 1 ml/mim, p = 0.01) without any change in blood pressure, followed by a decrease in TPR (1.4 ± 0.07 1.2 ± 0.1 (mmHg/ml/min). There was an increase in both diuresis and sodium excretion in P group. These alterations were followed by an increase in the mRNA levels for NHE3 (79%), BSC (100%) and AQP2 (250%) compared with C group. Similar to humans, the pregnant rats presented a rise in CO indicating an expansion of ECV with no changes in BP. The upreg-ulation of Na and water transporters in the kidney may be involved in ECV expansion during this especial physiological condition.
OF A NOVEL VASODILATOR PEPTIDE,
IN HUMAN KIDNEY
KAZUHIRO TAKAHASHI1, KUMI KIKUCHI2, TOMOKO URABE3, KIICHIRO NAKAJIMA3, HIRONOBU SASANO4,
KAZUHITO TOTSUNE5, OSAMU MURAKAMI2
Department of Molecular Biology & Applied Physiology, Tohoku University School of Medicine, Sendai, Japan1, Department of Medicine Tohoku University School of
Medicine2, Peptide Institute3, Department of Pathology4, Department of Clinical
Adrenomedullin 2/intermedin (AM2/IMD) is a novel member of the calci-tonin/calcitonin gene-related peptide (CGRP) peptide family, that was identified by searching the genome database. AM2/IMD has a vasodilator action, and anti-diuretic and antinatriuretic effects in mice. Immunocytochemical localization of AM2/IMD in human tissues has not been studied in detail. We studied expres-sion of AM2/IMD in human kidney as well as hypothalamus and heart by immunocytochemistry. Antiserum against human AM2/IMD was raised in a rabbit by injecting human AM2/IMD (1–7) conjugated with bovine thyroglob-ulin in the Peptide Institute Inc. (Osaka, Japan). In the human kidney obtained at autopsy, renal tubular cells were positively immunostained with AM2/IMD, whereas neither glomeruli nor vasculature in the kidney were immunostained. AM2/IMD-immunoreactive cell bodies are found in the paraventricular and
EFFECTS OF VANADATE AND LOW POTASSIUM
DIET ON RENAL NA,K-ATPASE AND H,K-ATPASE
PROTEIN EXPRESSION IN RAT
RATANAPORN JERAWATANA1, WIPAWEE KITTIKOWIT2, SOMCHAI EIAM-ONG3, SOMCHIT EIAM-ONG2
Inter-department of Physiology, Graduate School, Chulalongkorn University, Bangkok 10330, Thailand1, Department of Pathology, Faculty of Medicine,
Chulalongkorn University2, Department of Physiology, Department of Medicine,
Faculty of Medicine, Chulalongkorn University, Thailand3
Both vanadate and potassium depletion could alter renal Na,K-ATPase and H,K-ATPase activity. This study was conducted to investigate the effects of vanadate and low potassium diet on Na,K-ATPase and H,K-ATPase protein expression. Male Wistar rats were intraperitoneally injected with normal saline solution (NSS) or vanadate (V; 5 mg/kg. BW). Each group was received either normal-potassium (NK) or low-normal-potassium (LK) diet. The treatments were performed for 10 days. On experimental due date, 24-hr urine and blood samples were collected for measurement of vanadium, electrolytes, blood urea nitrogen, creatinine, and creatinine clearance. The kidneys were removed and determined for vanadium concentration, as well as were fixed for measurement of Na,K-ATPase and H,K-ATPase (alpha1 and alpha2 isoforms) protein expression. Vanadate administra-tion significantly increased vanadium levels in all parts studied in both NK and LK groups. By immunohistochemistry, the main staining of Na,K-ATPase protein expression was in distal tubule and collecting duct. The expression was increased by LK but reduced by V. LK in V treated rats could not restore the expression. For H,K-ATPase, in cortex, LK could enhance the expression of both alpha1 and alpha2 at luminal membrane of collecting duct. In medulla, LK slightly increased alpha1, whereas no expression of alpha2 protein was noted. Vanadate had no effect on both isoforms of H,K-ATPase expression. Treatment of V + LK caused a progressive hypokalemia, azotemia and natriuresis. LK with NSS or V signifi-cantly reduced blood pCO2 while blood pH was remained normal. Fractional excretion of potassium, chloride, and bicarbonate were increased in V + LK animal. Urine flow rate were comparable in all groups. These findings are the first evidence showing that LK could not restore the suppressive effect of V on renal Na,K-ATPase protein expression. Vanadate has no influence on renal H,K-ATPase protein expression. LK still plays an important stimulus on increasing of H,K-ATPase protein expression.
COLLECTRIN IS A NOVEL TARGET OF HNF-1 IN
RENAL COLLECTING DUCT CELLS
YANLING ZHANG1, JUN WADA1, AKIHIRO YASUHARA1, JUN EGUCHI1, IZUMI HASHIMOTO1, KAZUYA YAMAGATA2, KENICHI SHIKATA1, HIROFUMI MAKINO1
Okayama University Graduate School of Medicine and Dentistry1, Osaka
We identified the gene that encodes collectrin, a homologue of angiotensin-converting enzyme 2 (ACE2), which is upregulated in 5/6 ablated mouse kidneys at hypertrophic phase (Zhang H et al. Kidney Int, 1999; Zhang H et al. J Biol Chem, 2001). Collectrin, a novel member of ACE gene family, is membrane bound protein and exclusively localized and expressed in the collecting duct cells and mouse inner medulla collecting duct cells (mIMCD3). Collectrin lacks a cat-alytic domain, which indicates that there might be unknown functions besides simple peptide hydrolysis like ACE. Analysis of transcription factor binding sites in the promotor regions of human, rat, and mouse collectrin reveals the presence of hepatocyte nuclear factor-1 consensus binding sequences. We confirmed the specific binding of HNF-1a and HNF-1b to the putative HNF-1 binding site of collectrin gene by electrophoretic mobility shift assay (EMSA) in mIMCD3 cells. We subcloned a 322-bp of the 5¢ flanking promoter region of the human collec-trin gene into pGL3 Luciferase Reporter vector (Colleccollec-trin-pGL3) and co-transfected it with HNF-1a-pcDNA3.1(+) and/or HNF-1b-pcDNA3.1(+) into mIMCD3 cells and luciferase activity assays were performed using the Dual Luciferase Reporter Assay System. HNF-1a, HNF-1b, and co-transfection of HNF-1a and HNF-1b stimulated the promoter activity by 11.1 ± 1.9-, 2.4 ± 0.5-and 13.2 ± 0.7-fold compared with Collectrin-pGL3, respectively. We established stably expressing mIMCD3 cell lines by transfection with HNF-1a-pcDNA3.1(+) and selection in the presence of geneticin. By analyzing several cell lines, North-ern and WestNorth-ern blot analyses reveled that collectrin mRNA and protein was up-regulated in the HNF-1a highly expressing stable cell lines. These results suggest that HNF-1a and HNF-1b play important roles in the transcriptional regulation of collectrin and thus collectrin is a novel target of HNF-1 in collecting duct cells.
supraoptic nuclei of human hypothalamus. Both parvocellular and magnocellu-lar cells in the paravetricumagnocellu-lar nucleus are immunostained with AM2/IMD. Immunostaining of serial sections showed colocalization of AM2/IMD and vaso-pressin in these cells. Myocardial cells of the heart were positively immunos-tained. These results indicate that AM2/intermedin is expressed in the renal tubular cells, suggesting that this peptide may regulate water-electrolyte metab-olism and renal circulation in the kidney. Furthermore, expression in the hypo-thalamus and heart suggest that this novel peptide may be related to the central and peripheral regulation of the circulation and water-electrolyte metabolism.
IMCD WATER CHANNEL AQUAPORIN 2
EXPRESSION WAS INCREASED BY GLUCAGON IN
NORMAL RATSANTONIO-J MAGALDI1 , Y YANO1 , AC RODRIGUES1 , L C ANDRADE1
Clinical Hospital-University of São Paulo-LIM1
In animals supplied with a high protein diet the urinary concentrating capacity is enhanced and the release of Glucagon(Gl) is also increased. It is known that Gl participates in water excretion by the kidney after a protein meal and an i.v. amino acids infusion. However, the direct effect of this hormone on perfused (IMCD), is not well defined. Thus, the effect of Gl was examined in water trans-port in isolated IMCD perfused ‘in vitro’from normal rats. Osmotic Water Per-meability (Pfmm/s), was studied at 37ºC and pH 7.4 in normal rat IMCD(n = 26) perfused and bathed with Ringer/HCO3. Gl (10-7M) added to the bath fluid in absence of Vasopressin (Vp), enhanced the Pf from 4.38?.40 to 11.16?.44 (p < 0.01). Adding Gl 10-8, 10-7and 10-6M, the Pf responded in a dose-dependent manner. The Protein kinase A inhibitor H8 blocked the Gl effect. The specific Gl inhibitor, des-His1-[Glu9] glucagon (10-7M), blocked the Gl stim-ulated Pf but not the Vasopressin(Vp) stimstim-ulated Pf. The cAMP level enhanced from the control 1.24?.39 fm/mg prot to 59.70?5.18 after Gl 10-7M in an IMCD tubule suspension. These results are in agreement with the subsequent immunobloting studies that indicated an increase in AQP2 protein abundance of 27% compared with the controls(cont-100.0?3.9 vs Gl 127.53, p = 0.0035) in membrane fraction extracted from IMCD tubule suspension, incubated with 10-6M Gl. Our data showed that: 1- Gl increased water absorption in a dose-dependent manner; 2- The anti-Gl blocked the action of Gl but not the action of Vp; 3- Gl stimulated the cAMP generation; 4- Gl increased the AQP2 water channel protein expression, leading us to conclude that Gl affects water absorp-tion by utilizing a Gl receptor(rather than a Vp receptor) increasing the AQP2 protein expression.
THE ROLE OF RENAL DOPAMINE SYSTEM IN
SODIUM RETENTION OF PAN AND
BENEDITA SAMPAIO-MAIA1, M MOREIRA-RODRIGUES2, O AZEVEDO-SILVA2, P SERRAO1, M PESTANA2
Inst Pharmacology and Therapeutics1, Unit of Research and Development of
Nephrology, Faculty of Medicine, Porto, Portugal2
This study evaluated the role of renal dopamine in the sodium retention of puromycin aminonucleoside (PAN) and HgCl2-induced nephrosis. Sprague-Dawley rats (Harlan, Spain) weighing 150 g received PAN (150 mg/kg bw, ip; n = 9) or the vehicle (0.9% NaCl, ip; n = 9) on day 0. Brown-Norway rats (Harlan, Spain) weighing 150 g received HgCl2(1 mg/kg bw, sc; n = 9) or the vehicle (0.9% NaCl, sc; n = 9) on days 0, 2, 4, 7, 9 and 11. Twenty-four hours urine was collected for determination of sodium and dopamine. The renal aromatic L-amino acid decarboxylase activity (AADC), the enzyme responsible for dopamine synthesis, was evaluated in PAN-treated rats on days 7 and 14 and in HgCl2-treated rats on days 7, 14 and 21. During greatest sodium retention and ascites accumulation (day 7 for PAN-treated rats and day 14 for HgCl2-treated animals) the nephrotic and control rats were submitted to a 5% bw volume expansion (VE) with saline and the effects of the D1agonist (fenoldopam, 10 mg/kg bw/min) on the natriuresis and on the Na+,K+-ATPase activity in the renal proximal tubules was evaluated. A marked decrease in renal AADC activity was observed in PAN and HgCl2-treated rats on days 7, 14 and 21. This was accom-panied in PAN and HgCl2-treated rats with a marked decrease in the daily urinary excretion of dopamine. The VE was accompanied with a blunted natriuretic response in both PAN and HgCl2-treated rats. During fenoldopam infusion the natriuretic response to VE was significantly increased in control rats but was not
altered in both PAN and HgCl2-treated animals. The fenoldopam-induced inhi-bition of Na+,K+-ATPase activity in renal proximal tubules was well preserved in either PAN or HgCl2-treated rats. We conclude that a blunted renal dopamin-ergic tonus is observed in both PAN and HgCl2-induced nephrosis. This may contribute to increase the proximal sodium retention in a state of proteinuria. (Grant-POCTI/FCB/45660/2002-FCT)
TUBULAR TRANSPORTERS AND CLEARANCE
SERVAIS A1, LECHAT P1, ZAHR N1, URIEN S1, AYMARD G1, JAUDON MC1, DERAY G1, ISNARD BAGNIS C1
Pitié-Salpêtrière University Hospital1
Introduction: Adefovir was shown to be effective in hepatitis B virus therapy.
It is transported in vitro by two tubular transporters, the organic anion transporter (OAT1) and the multidrug resistant protein (MRP2). We studied adefovir clear-ance in vivo in rat after pharmacological inhibition of transporters by probenecid and in mutant transport-deficient rats (TR-), in which MRP2 is lacking.
Methods: After treatment by probenecid or placebo, adefovir (10 or 30 mg/kg)
was administered via tail artery to normal Wistar rats or to TR- rats. Sequential blood and urine samples were collected during 48 hours and pharmacokinetics was studied via population modeling (NONMEM).
Results: The fraction of drug excreted in the urine was low (table 1). Renal
clearance of adefovir in control TR- rats (0.13 +/- 0.07 l/h) was not different from normal control but it was significantly lower (p < 0.05) in probenecid TR-rats (0.03 +/- 0.02 l/h) than in normal control (0.09 +/- 0.05 l/h), in normal probenecid (0.10 +/- 0.07 l/h) and in TR- control rats (0.13 +/- 0.07 l/h). Non renal clearance of adefovir 10 mg/kg increased significantly in probenecid normal rats (0.47 +/- 0.10 l/h) compared to normal controls (0.29 +/- 0.11 l/h, p < 0.0001).
Conclusion: In vivo in rats MRP2 mutation alone did not affect adefovir
clear-ance suggesting that MRP2 does not play a critical role in the secretion of adefovir. Additional pharmacological inhibition of transporters decreased renal clearance which may reflect inhibition of compensating transport mechanisms activated when MRP2 is lacking. Drug interactions on these transporters may affect efficiency and toxicity of adefovir.
ALTERED REGULATION OF WATER AND SODIUM
TRANSPORT SYSTEMS IN THE KIDNEY OF RATS
TREATED WITH GLYCYRRHIZIC ACID
KI CHUL CHOI1, SEONG KWON MA1, YOUN KYOUNG LEE1, SOO WAN KIM1, NAM HO KIM1, JONGUN LEE2
Department of Internal Medicine, Chonnam National University Medical School1,
Department of Physiology, Chonnam National University Medical School2
The present study was aimed to determine whether there exists an altered regu-lation of aquaporin (AQP) water channels and sodium transport systems in the kidney in licorice-induced hypertension. Male Sprague-Dawley rats were treated with glycyrrhizic acid (GA) in drinking water (200 mg/dL) for 3 weeks. Systolic blood pressure (SBP) was measured by the tail-cuff method. Plasma renin activ-ity (PRA), serum aldosterone levels, and plasma concentrations of arginine vaso-pressin (AVP) were determined by radioimmunoassay. The protein expressions of AQP1-3, heteromeric G protein subunit (Gsa) and Na,K-ATPase a1 subunit were determined in the kidneys by Western blot analysis. The enzymatic activ-ity of Na,K-ATPase was also determined. SBP increased significantly following the GA treatment (129.7 ± 0.9 vs. 153.0 ± 4.1 mmHg, p < 0.01, n = 10 each). Accordingly, PRA was significantly decreased (3.7 ± 1.0 vs. 1.2 ± 0.6 ng/mL/hr, p < 0.05, n = 6 each). Serum aldosterone levels were also decreased significantly (19.6 ± 2.5 vs. 5.3 ± 0.8 ng/dL, p < 0.05, n = 6 each). Levels of plasma AVP did not differ between the groups. In the GA group, the expression of AQP1 was not significantly altered. The expression of AQP2 was significantly increased in the outer medulla and inner medulla, while unaltered in the cortex. The expression of AQP3 was also increased in the outer medulla and inner medulla, while unal-tered in the cortex. The expression of Gsa was increased in the inner medulla, while unaltered in the cortex and outer medulla. The enzymatic activity of Na,K-ATPase was significantly increased in the kidney, and the protein expression of Na,K-ATPase a1 subunit was increased in the cortex of the kidney. The increased expression of AQP2–3 and the up-regulation of Na,K-ATPase may play a role in hypertension following the ingestion of glycyrrhizic acid.
EFFECTS OF FUROSEMIDE ON GENE EXPRESSION
OF RENAL CALCIUM AND MAGNESIUM
CHIEN-TE LEE1, HUNG-CHUN CHEN2, KIM-CHONG YONG3, LI-WEN LAI3, YEOUNG-HAU LIEN3
Chang-Gung Memorial Hospital, Kaohsiung, Taiwan1, Graduate Institute of
Medicine, Kaohsiung Medical University2, Department of Medicine, Health Sciences
Center, University of Arizona, USA3
Loop diuretics are effective agents in treating edema and hypertension. In addi-tion to diuresis and natriuresis, increased urinary calcium (Ca) and magnesium (Mg) excretion are often observed. The pathomechanism of the enhanced Ca and Mg excretion is direct inhibition on reabsorption in thick ascending limb of Henle. As the distal convoluted tubule (DCT) plays an important role in Ca and Mg transport, we aim to evaluate the alternations in renal Ca and Mg transport in DCT after furosemide treatment. In acute experiments, the mice were admin-istered with single dose furosemide (10 mg/kg/dose), and for chronic experiments, twice injections with same dosage were given for 3 days. Salt drinking water was given as supplement to prevent diuretics-induced volume depletion in chronic experiment. Serum creatinine (Cr), Ca and Mg were determined as well as urine samples. Quantitative analysis of gene expression was performed using real-time RT-PCR. Our results showed that urinary Ca and Mg excretion were increased after single injection (urinary Ca/Cr: 0.09 ± 0.11 vs. 0.18 ± 0.02, Mg/Cr: 0.48 ± 0.14 vs. 1.31 ± 0.27, both p < 0.05). Significant increase in gene expression of TRPV5 (174 ± 10% of control) and calbindin-D28k (222 ± 23%) were found. Chronic treatment also induced calciuresis and magnesiuria with or without salt supplement (Ca/Cr: 0.26 ± 0.17, 0.32 ± 0.14; Mg/Cr: 0.96 ± 0.1, 0.98 ± 0.1, all p < 0.05). This effect was associated with increased expression of Ca and Mg chan-nels and calbindins (TRPV5: 170 ± 10%, 212 ± 15%; TRPM6: 230 ± 11%, 293 ± 19%; calbindin-D28k: 310 ± 18%, 290 ± 21%; calbindin-D9k: 220 ± 20%, 255 ± 18%, all p < 0.05). We conclude that furosemide treatment causes significant renal Ca and Mg loss. Salt supplement did not correct the renal wasting. Up-regulation of Ca and Mg transport molecules in DCT represent an adaptive response to increased Ca and Mg delivery induced by furosemide.
ANION TRANSPORTERS SLC26A6 AND
SLC26A7 HAVE SPECIFIC EXPRESSION IN
JUXTAGLOMERULAR APPARATUS OF
MINNA KUJALA1, JUKKA TIENARI2, HANNES LOHI3, HANNU SARIOLA4, EERO LEHTONEN5, JUHA KERE6
Department of Medical Genetics, University of Helsinki, and Department of Urology, Helsinki University Central Hospital, Finland1, Department of Pathology,
Helsinki University Central Hospital/Peijas Hospital, Finland2, Department of
Medical Genetics, University of Helsinki, Finland3, Institute of Biomedicine,
Developmental Biology, University of Helsinki, and HUCH Laboratory Diagnostics, Helsinki University Hospital, Finland4, Department of Pathology, Haartman
Institute and Helsinki University Central Hospital, University of Helsinki, Finland, and Department of Cellular and Molecular Medicine, University of California at San Diego, La Jolla, CA, USA5, Department of Medical Genetics, University of
Helsinki, Finland, and Department of Biosciences at Novum, Karolinska Institutet, Stockholm, Sweden6
Background: The solute carrier protein family 26 (SLC26), formerly known as
sulphate transporters, consists of membrane proteins all of which but one transport anions with different specificities. Two of them, SLC26A6 (PAT1) and SLC26A7, are expressed in kidney, but their exact localization in human kidney has not been reported. Both proteins transport at least chloride, oxalate, sulphate and bicarbonate. Interestingly, expression and function of at least some of the orthologous SLC26 proteins vary between different mammalian species. We therefore studied expression of SLC26A6 and A7 in human kidney.
Methods: Localization of SLC26A6 and A7 in human kidney was studied by
RT-PCR, Western blotting and immunohistochemistry.
Results: In addition to expression in specific tubule segments, both SLC26A6
and A7 are localized to the juxtaglomerular apparatus. SLC26A6 is expressed in the macula densa (MD) cells, while SLC26A7 is localized to the extraglomeru-lar mesangial cells (EMC) in human kidney. To our knowledge, expression of these anion transporters has never before been reported in the juxtaglomerular apparatus of any species.
Discussion: Both MD and EMC are important in the tubuloglomerular feedback
(TGF) involved in the autoregulation of glomerular filtration and renal blood flow. MD functions as the sensor of the luminal Na + and Cl- concentrations.
INFLUENCE OF ANTISENSE CUBILIN RNA
ON ALBUMIN-INDUCED EXPRESSIONS OF
CHEMOKINS IN HUMAN RENAL PROXIMAL
TUBULAR EPITHELIAL CELLS (HK-2)JURONG YANG1, YU HY1, LIN JING1, HE YN1 Dept. of Nephrology, Daping Hospital of TMMU1
Objective: To investigate the role of cubilin on albumin-induced expressions of
monocyte chemoattractant protein 1 (MCP-1) and regulated upon activation normal T-cell expressed and secreted (RANTES) in human renal proximal tubular epithelial cells (HK-2).
Methods: The sense and antisense cubilin recombinant eukaryotic expression
vectors (pcDNA-CUB and pcDNA-ACUB) were constructed using gene recom-bining techniques and identified by restriction endonucleases digesting and DNA sequencing. Both vectors as well as pcDNA3.1(+) vector were respectively trans-fected into HK-2 cells with lipofectin DOTAP. Flow cytometer (FCM) was applied to determine the inhibitive effect of antisense cubilin RNA on albumin uptake of HK-2 cells. Posterior to cell transfection for 72 hours, high concen-tration of albumin (10 g/L) was used for stimulation for 24 hours. The expres-sions of MCP-1 and RANTES in HK-2 cells were measured by ELISA and Western blot.
Results: Compared with DOTAP group, the albumin uptake was significantly
lowered (p < 0.01) and both MCP-1 and RANTES expressions of HK-2 cells sig-nificantly decreased in pcDNA-ACUB trasnfection group (p < 0.001).
Conclusion: Antisense cubilin RNA can inhibit albumin uptake of HK-2 cells
and suppress the albumin-induced up-regulation of MCP-1 and RANTES expres-sion in the HK-2 cells, as indicates that cubilin may play an important role in albumin-induced chemokines expression in renal proximal tubular epithelial cells.
SYMPATHETIC REGULATION OF AQUAPORIN-2
WATER CHANNELS IN THE KIDNEY IN RATSKI CHUL CHOI1, YOONWHA OH2, YOUN KYOUNG LEE1, SEONG KWON MA1, SOO WAN KIM1, NAM HO KIM1, JONGUN LEE2 Department of Internal Medicine, Chonnam National University Medical School1,
Department of Physiology, Chonnam National University Medical School2
Whether there exists a sympathetic neural mechanism in the regulation of aqua-porin-2 (AQP2) water channels in the kidney was examined. Male Sprague-Dawley rats were used. Four different series of experiments were done. In the first series, the renal nerve was unilaterally denervated by stripping the nervous and connective tissues passing to and along the course of the renal artery and vein and painting these vessels with a solution of 10% phenol through a midline abdominal incision. They were kept for 3 days until sacrificed. In another series of experiment, chemical sympathectomy was achieved by intravenous injection of 6-hydroxydopamine (1 mg/kg). They were used 2 days later. In the third series, rats were unilaterally renal nerve-denervated and subjected to a water restriction. During the days of water restriction, the rats were intraperitoneally infused of V2 receptor antagonists (arginine vasopressin fragment 4–9, 5 mg/kg/day) through osmotic minipump. In the last series, deoxycorticosterone acetate (DOCA)-salt hypertension was induced in renal nerve-intact and denervated rats. The expres-sion of AQP2 proteins was determined in the kidneys. Following the denerva-tion, the norepinephrine content in the denervated kidney was significantly decreased, while that in the contralateral kidney increased. Accordingly, the expression of AQP2 proteins was markedly decreased in the denervated kidney. The chemical sympathectomy with 6-hydroxydopamine markedly decreased the expression of AQP2 proteins in the kidney. Following the water-restriction, the expression of AQP2 channels increased, however, the magnitude of which was lower in the denervated than in the contralateral innervated kidney. The renal nerve denervation significantly attenuated the development of DOCA-salt hypertension, along with a less increased expression of AQP2 proteins in the kidney. It is suggested that the sympathetic nervous system has a tonic stimula-tory effect on the expression of AQP2 water channels in the kidney.