Reduced LRP6 expression and increase in the interaction of GSK3ß with p53 contribute to podocyte apoptosis in diabetes mellitus and are prevented by green tea.

In diabetes mellitus (DM), podocyte apoptosis leads to albuminuria and nephropathy progression. Low-density lipoprotein receptor-related protein 6 (LRP6) is WNT pathway receptor that is involved in podocyte death, adhesion and motility. Glycogen synthase kinase 3 (GSK3) interaction with p53 (GSK3-p53) promotes apoptosis in carcinoma cells. It is unknown if GSK3-p53 contributes to podocyte apoptosis in DM. In experimental DM, green tea (GT) reduces albuminuria by an unknown mechanism. In the present study, we assessed the role of the GSK3ß-p53 in podocyte apoptosis and the effects of GT on these abnormalities. In diabetic spontaneously hypertensive rats (SHRs), GT prevents podocyte's p-LRP6 expression reduction, increased GSK3ß-p53 and high p53 levels. In diabetic SHR rats, GT reduces podocyte apoptosis, foot process effacement and albuminuria. In immortalized mouse podocytes (iMPs), high glucose (HG), silencing RNA (siRNA) or blocking LRP6 (DKK-1) reduced p-LRP6 expression, leading to high GSK3ß-p53, p53 expression, apoptosis and increased albumin influx. GSK3ß blockade by BIO reduced GSK3ß-p53 and podocyte apoptosis. In iMPs under HG, GT reduced apoptosis and the albumin influx by blocking GSK3ß-p53 following the rise in p-LRP6 expression. These effects of GT were prevented by LRP6 siRNA or DKK-1. In conclusion, in DM, WNT inhibition, via LRP6, increases GSK3ß-p53 and podocyte apoptosis. Maneuvers that inactivate GSK3ß-p53, such as GT, may be renoprotective in DM.

Role of caspases on cell death, inflammation, and cell cycle in glycerol-induced acute renal failure.

Caspases are the main executioners of apoptosis as well as interleukin (IL)-1beta and IL-18 conversion to active forms. They are activated after acute kidney injuries. In this study, we evaluated the importance of the caspase family in the pathogenesis and recovery of glycerol-induced acute renal failure in rats (Gly-ARF). Rats were treated with pan-caspase or selective caspase 1 and 3 inhibitors at the moment we injected glycerol. Renal function, renal histology (HE), transferase-mediated deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling staining for apoptosis, leukocytes infiltration (immunohistochemistry), renal expression of IL-1beta and IL-18 (immunohistochemistry and Western blot), tubular regeneration (5-bromo-2'-deoxyuridine (BrdU) incorporation), and P27(Kip) expression (Western blot) were evaluated at appropriate times. All inhibitors reduced the renal function impairment. Pan-caspase and caspase-3 inhibitors reduced cellular death (necrosis and apoptosis) 24 h after Gly-ARF. All caspases inhibitors reduced macrophages infiltration. The expression of total IL-1beta was enhanced in Gly-ARF, but the active IL-1beta and IL-18 forms were abolished in pan-caspase treated rats. Caspase-1 inhibitor attenuated Gly-ARF but not tubular injury suggesting glomerular hemodynamic improvement. There was striking regenerative response 48 h after Gly-ARF characterized by enhanced BrdU incorporation and reduced expression of p27(Kip). This response was not blunted by caspases inhibition. Our findings demonstrate that caspases participate in important pathogenic mechanisms in Gly-ARF such as inflammation, apoptosis, vasoconstriction, and tubular necrosis. The early inhibition of caspases attenuates these mechanisms and reduces the renal function impairment in Gly-ARF.

Prevention of hypertension attenuates albuminuria and renal expression of fibronectin in diabetic spontaneously hypertensive rats.

BACKGROUND/AIM: The aim of this study was to evaluate the effect of preventing hypertension on renal disease in a model of genetic hypertension and diabetes. METHODS: Four-week-old spontaneously hypertensive rats (SHR) with streptozotocin-induced diabetes were randomized for no treatment, or for treatment with captopril, losartan or triple therapy (hydrochlorothiazide, reserpine and hydralazine) for 16 weeks. RESULTS: Increase in systolic blood pressure was equally prevented by captopril (128 +/- 3 mm Hg), losartan (128 +/- 2) and triple therapy (129 +/- 2, p < 0.0001). Albuminuria was similarly reduced by captopril (499 (404-659)), losartan (622 (470-976)) and triple therapy (479 (362-600) microg/24 h (p < 0.0001)). Renal fibronectin expression increased in diabetic SHR (125 +/- 13 densitometric unit) as compared to the controls (51 +/- 9, p < 0.0001), and decreased (p < 0.0001 vs. diabetic SHR) with captopril (32 +/- 8), losartan (27 +/- 4) and triple therapy (35 +/- 6). CONCLUSION: The prevention of hypertension in diabetic SHR by captopril, losartan or triple therapy was equally efficacious in impeding increase of albuminuria and the expression of renal fibronectin. Under these conditions, tight blood pressure control was the main determinant in attenuating nephropathy.

The presence of genetic hypertension stimulates early renal accumulation of fibronectin in experimental diabetes mellitus.

AIMS/HYPOTHESIS: To investigate the interaction between hypertension and diabetic nephropathy, we studied the renal accumulation of fibronectin in a genetic model of hypertension with streptozotocin-induced diabetes mellitus. METHODS: Spontaneously hypertensive rats (SHR) and their genetically normotensive control Wistar Kyoto rats (WKY) were studied at 4 weeks of age. The rats were killed 20 days after the induction of diabetes mellitus. The renal accumulation of fibronectin was estimated using Western blot analysis as well as immunofluorescence technique and confocal microscopy. RESULTS: Blood glucose concentrations were similar in diabetic SHR rats (27 +/- 3.3 mmol/l) and WKY rats (25.5 +/- 2.7 mmol/l). The systolic blood pressure was higher in both groups of SHR rats than in the control rats. The abundance of renal fibronectin as detected by Western blot analysis was (p < 0.05) higher in the diabetic SHR rats (41.4 +/- 15.0 densitometric U, n = 8) than in the control rats, and no difference was observed between diabetic and control WKY rats (20.8 +/- 6.2, n = 8) and (27.8 +/- 17.2, n = 8). The mean peak intensity of fibronectin signal within the glomerulus as estimated by confocal microscopy was higher (p < 0.05) in the diabetic SHR rats (32.9 +/- 3.5) than in control SHR rats (11.9 +/- 5.7) or diabetic (7.4 +/- 2.2) and control (15.2 +/- 7.9) WKY rats. CONCLUSION/INTERPRETATION: In experimental diabetes the presence of genetic hypertension promotes earlier accumulation of renal fibronectin, a matrix protein implicated in renal glomerulosclerosis.

Erythrocyte sodium-lithium countertransport and proliferative diabetic retinopathy.

PURPOSE: To investigate whether elevated erythrocyte Na+/Li+ countertransport (Na+/Li+ CT) activity is present in patients with proliferative diabetic retinopathy (PDR). METHODS: The rate of Na+/Li+ CT activity assayed in 21 patients with type 1 diabetes mellitus (DM) presenting PDR was compared with 10 patients with nonproliferative retinopathy (NPDR) and with 11 patients with normal fundi. Twelve normal volunteers with no family history of hypertension were used as a control group. The albumin excretion rate was determined by nephelometry, and the glomerular filtration rate was measured by the plasma clearance of eidetic acid labeled with chromium-51. RESULTS: Patients with PDR showed higher diastolic blood pressure levels (mean +/- SD) compared with those with NPDR or normal fundi (95 +/- 13 versus 90 +/- 09 and 82 +/- 19 mm Hg, P = 0.02, respectively). The albumin excretion rate was higher [geometric mean (range)], and the glomerular filtration rate was lower (mean +/- SD) in patients with PDR than in those with NPDR or normal fundi [333 (2 to 5140) versus 32 (5.9 to 2200) and 6 (1.5 to 306) microg/min, P = 0.01, and 63 +/- 33 versus 99 +/- 37 and 93 +/- 43 ml/min, P = 0.02, respectively]. The mean Na+/Li+ CT in patients with PDR was significantly higher than in patients with NPDR or normal fundi and control group (0.46 +/-0.20 versus 0.32 +/- 0.12, 0.32 +/- 11, and 0.21 +/- 0.07 mM/L red blood cells (RBC)/h, respectively, P = 0.0001). In a multiple logistic regression analysis, with PDR as the dependent variable, Na+/Li+ CT (odds ratio [OR]: 4.7, confidence interval [CI]: 1.2-17.6, P = 0.02), diastolic blood pressure (OR, 3.4; CI, 1.3 to 9.6; P = 0.018), and glomerular filtration rate (OR, 5.1; CI, 1.6-17.7; P = 0.007) were the only variables that were maintained in the equation, indicating that they were the main determinants of PDR. CONCLUSIONS: Patients with type 1 DM and proliferative retinopathy have elevated erythrocyte Na+/Li+ CT.

Renal haemodynamic responses to a chicken or beef meal in normal individuals.

BACKGROUND: In normal subjects, protein loading with soybean meal does not produce the same renal haemodynamic effects as those observed with a beef meal. The renal responses of an acute protein load in the form of chicken meal is unknown. METHODS: To examine whether the renal response to a chicken meal differs from that to beef, we studied the renal function of eight normal healthy volunteers before and after a protein load with each of these meals. In a crossover randomized study, we measured the glomerular filtration rate (GFR; inulin clearance), renal plasma flow (RPF; para-aminohippurate clearance) and, plasma amino acid and glucagon levels. We also determined the amino acid content of a sample of chicken and beef. RESULTS: GFR and RPF increased significantly 2 h after both the chicken and beef meals (chicken, 98+/-13 vs 119+/-18 and 476+/-123 vs 570+/-99 ml/min/1.73 m2; beef, 107+/-14 vs 122+/-16 and 501+/-118 vs 560+/-97 ml/min/1.73 m2, for GFR and RPF at basal and 2 h respectively, P<0.05). Renal vascular resistance decreased and the filtration fraction remained unchanged after both protein loads. The changes induced by the protein challenges in the plasma amino acid and glucagon levels were not different between the two protein sources. The amino acid contents of chicken and beef samples were similar. CONCLUSION: In normal subjects, chicken and beef meals induced a similar degree of hyperfiltration.

Band 3 Campinas: a novel splicing mutation in the band 3 gene (AE1) associated with hereditary spherocytosis, hyperactivity of Na+/Li+ countertransport and an abnormal renal bicarbonate handling.

We have studied the molecular defect underlying band 3 deficiency in one family with hereditary spherocytosis using nonradioactive single strand conformation polymorphism of polymerase chain reaction (PCR) amplified genomic DNA of the AE1 gene. By direct sequencing, a single base substitution in the splicing donor site of intron 8 (position + 1G --> T) was identified. The study of the cDNA showed a skipping of exon 8. This exon skipping event is responsible for a frameshift leading to a premature stop codon 13 amino acids downstream. The distal urinary acidification test by furosemide was performed to verify the consequences of the band 3 deficiency in alpha intercalated cortical collecting duct cells (alphaICCDC). We found an increased basal urinary bicarbonate excretion, associated with an increased basal urinary pH and an efficient distal urinary acidification. We also tested the consequences of band 3 deficiency on the Na+/H+ exchanger, by the measurement of Na+/Li+ countertransport activity in red blood cells. The Na+/Li+ countertransport activity was increased threefold to sixfold in the patients compared with the controls. It is possible that band 3 deficiency in the kidney leads to a decrease in the reabsorption of HCO3- in alphaICCDC and anion loss, which might be associated with an increased sodium-lithium countertransport activity.

Mesangial cell abnormalities in spontaneously hypertensive rats before the onset of hypertension.

To identify kidney biosynthetic abnormalities that may precede the onset of hypertension, we studied the expression of fibronectin (FN) and collagen IV (Coll IV) in young SHR (4 weeks of age) whose systolic blood pressure was normal and similar to that of age-matched control WKY rats. In isolated glomeruli the level of FN protein assessed by immunoblotting tended to be lower in the SHR than in the WKY rats. By Northern analysis the FN/actin mRNA ratio was significantly lower in glomeruli from SHR (0.56 +/- 0.47) than in glomeruli from WKY rats (2.0 +/- 0.8). These abnormalities were maintained in vitro since the expression of FN was significantly lower in SHR than in WKY cultured mesangial cells (FN/actin mRNA ratio = 0.84 +/- 0.46 vs. 1.9 +/- 0.7, P = 0.029). No differences in Coll IV mRNA or protein levels were observed in SHR glomeruli and mesangial cells when compared with WKY rats. The levels of aortic FN and Coll IV mRNAs were not different in SHR and WKY rats. In addition, mesangial cells from SHR showed a significantly higher growth rate than those from WKY. The biosynthetic and proliferative abnormalities observed in the SHR mesangial cells appear to reflect genetic characteristics, and could provide novel insights into cellular mechanisms linking the genetics of hypertension with predisposition to glomerular pathology.

Renal functional response to protein loading in type 1 (insulin-dependent) diabetic patients on normal or high salt intake.

Insulin-dependent diabetes mellitus (IDDM) patients may have an increased intrarenal angiotensin II activity. In diabetic patients, captopril increases the renal hemodynamic response to an amino acid infusion. We investigated the effects of two salt diets on arterial pressure and renal response to a protein load in 10 normotensive (blood pressure < 140/90 mm Hg) IDDM patients (aged 30 +/- 3 years) who had diabetes for 7 +/- 4 years and normoalbuminuria levels [albumin excretion rate 4.8 (2.5-19.1) microg/min]. After 1 week of normal (approximately 100 mmol/day; approximately 100 mEq/l) and 1 week of high (approximately 300 mmol/day; approximately 300 mEq/l) salt intake, renal hemodynamic studies were performed at baseline and after a protein load (meat meal) of 100 g/1.73 m2. The mean 24-hour urinary sodium excretion levels were 99 +/- 27 and 293 +/- 80 mmol (mEq) with normal and high salt intake, respectively. No significant changes were seen in plasma sodium and glucose control with the normal and high salt diets, respectively: plasma sodium 135 +/- 3 vs. 137 +/- 1 mmol/l (mEq/l), (p = 0.08) and glycated hemoglobin 9.1 +/- 1.9 vs. 9.4 +/- 2.1% (p = 0.36). The body weight (70.9 +/- 12 vs. 71.8 +/- 13 kg; p = 0.015) was significantly higher with a high salt diet. The mean arterial pressure was similar with both diets (normal vs. high salt diet 91 +/- 9 vs. 89 +/- 6 mm Hg, p = 0.25). The plasma renin concentration [28 +/- 15 vs. 16 +/- 6 microU/ml(168 +/- 90 vs. 96 +/- 36 pmol/l), p = 0.013] and angiotensin II [8.8 +/- 4.4 vs. 6.4 +/- 3.5 pg/ml (0.052 +/- 0.025 vs. 0.038 +/- 0.021 nmol/l), p = 0.016] were significantly lower with the high salt diet. Following protein loading, the glomerular filtration rate increased with both diets: normal salt diet 114 +/- 26 vs. 128 +/- 30 ml/min/1.73 m2(1.9 +/- 0.43 vs. 2.13 +/- 0.50 ml/s/1.73 m2), p = 0.04; high salt diet 118 +/- 23 vs. 127 +/- 29 ml/min/1.73 m2 (1.97 +/- 0.38 vs. 2.12 +/- 0.48 ml/s/1.73 m2), p = 0.13. The change in renal plasma flow was similar to that of the glomerular filtration rate with normal and high salt intake, respectively: 566 +/- 94 vs. 617 +/- 142 ml/min/1.73 m2 (9.44 +/- 1.57 vs. 10.29 +/- 2.37 ml/s/173 m2), p = 0.0017; 572 +/- 125 vs. 600 +/- 110 ml/min/1.73 m2 (9.54 +/- 2.08 vs. 10.00 +/- 1.83 ml/s/1.73 m2), p = 0.057. In this subset of normotensive normoalbuminuric IDDM patients, a high salt intake did not promote an exaggerated renal response to the protein load despite inhibition of the renin-angiotensin system.

[Prevalence of diabetic nephropathy in adult patients with chronic kidney failure]. / Prevalência da nefropatia diabética em pacientes adultos com insuficiência renal crônica terminal.

UNLABELLED: Diabetic nephropathy is a frequent cause of end-stage renal failure in patients admitted for renal replacement therapy. PURPOSE: To evaluate the prevalence of DN, as the underline disease, in patients with ESRF. METHODS: 1,303 [male (M) = 767 and female (F) = 536] patients with ESRF who were on a waiting list for cadaver kidney transplant at Nephrology Unit-University Hospital (HC-UNICAMP), from August/90 to June/93--group 1--and 193 (M = 112 and F = 81) patients admitted for renal replacement therapy in a year period (April/92 to March/93), in the city of Campinas, State of São Paulo, Brazil, were studied. RESULTS: The prevalence of DN was 10.1% in group 1 and 17.6% in group 2 (x2 = 7.15; p = 0.007), being the third cause of ESRF in both groups, and it was preceded by glomerulonephritis and arterial hypertension. In group 1 the reduction of number of patients with increase in duration of dialysis was significantly greater in patients with diabetic nephropathy (x2 = 30.9; p < 0.001). Among patients with DN 35 (26%) in group 1 and 6 (18%) in group 2 had less than 35 years when they were admitted for renal replacement therapy and are likely to be type 1 (insulin-dependent) diabetic patients. CONCLUSION: In our studied groups DN was a frequent cause of ESRF.

Sodium-lithium countertransport activity and insulin resistance in normotensive IDDM patients.

In insulin-dependent diabetes (IDDM), an overactivity of sodium-lithium countertransport (Na+/Li+ CT) has been associated with the risk of nephropathy and hypertension, two conditions of insulin resistance. We investigated the sensitivity to insulin with a hyperinsulinemic (approximately 719 pM [approximately 100 microU/ml]) euglycemic clamp in two groups of normotensive nonproteinuric IDDM patients; 12 (10 men, 2 women) had high Na+/Li+ CT activity (mean 0.47, range 0.42-0.68 mmol/L red blood cells [RBC]/h, group 1) and 12 (9 men, 3 women) had normal Na+/Li+ CT activity (mean 0.24, range 0.12-0.31 mmol/L RBC/h, group 2). The two groups were similar in age (mean +/- SE 36 +/- 2 vs. 33 +/- 1 yr), duration of diabetes (19 +/- 3 vs. 18 +/- 2 yr), body mass index (26 +/- 0.8 vs. 24 +/- 0.6 kg/m2), arterial blood pressure (systolic/diastolic 121 +/- 4/79 +/- 2 vs. 122 +/- 3/77 +/- 2 mmHg), and glycemic control (HbA1 8.5 +/- 0.4 vs. 8.0 +/- 0.4%). Albumin excretion rate (AER) ranged between 4.7 and 148 (geometric mean 14) micrograms/min in group 1 and between 2.7 and 93 (geometric mean 11) micrograms/min in group 2. There were four microalbuminuric patients (AER greater than 30 micrograms/min) in each group. Whole-body glucose uptake was significantly reduced on average in group 1 compared with group 2 (41.6 +/- 2.2 mumol.kg-1.min-1 [7.48 +/- 0.4 mg.kg-1.min-1] vs. 49.6 +/- 2.2 mumol.kg-1.min-1 [8.93 +/- 0.4 mg.kg-1.min-1, P = 0.03), but some overlap existed between the two groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Prevalence of raised sodium-lithium countertransport activity in type 1 diabetic patients.

The prevalence of raised Na+/Li+ countertransport (CT) activity (greater than 0.41 mmol/liter RBC/hr) was assessed in 185 consecutive insulin-dependent diabetic patients attending an outpatient diabetic clinic. Normoalbuminuria was defined as an overnight albumin excretion rate (AER) of less than 20 micrograms/min (N = 121), microalbuminuria as AER between 20 and 150 micrograms/min (N = 35) and macroalbuminuria as AER greater than or equal to 150 micrograms/min (N = 29). The prevalence of elevated Na+/Li+CT (greater than 0.41 mmol/liter RBC/hr) was 21.5, 42.8 and 51.7% (P = 0.0005), in patients with normo-, micro- and macroalbuminuria, respectively. In the whole group, Na+/Li+CT was significantly related to mean blood pressure (MBP; rs = 0.37, P less than 0.001) and AER (rs = 0.38, P less than 0.001). In a multiple regression analysis the significant correlates of AER, as a continuous variable, or of proteinuria (micro + macroalbuminuria), as a categorical variable, were Na+/Li+CT, MBP, duration of diabetes and glycosylated hemoglobin (HbA1). The frequency of normoalbuminuric patients with high Na+/Li+CT activity fell with duration of diabetes. The risk of proteinuria was significantly greater in patients with raised Na+/Li+CT compared to those with Na+/Li+CT within the normal range (odds ratio 3.8, 95% CI, 1.9 and 7.8). A relative excess of patients with proteinuria (micro + macroalbuminuria) was found in the group with elevated Na+/Li+CT and HbA1 above the median value (8.05%) of the whole population (chi 2 = 9.7, P less than 0.002).(ABSTRACT TRUNCATED AT 250 WORDS)

[Nephrotic syndrome caused by membranous glomerulonephritis associated with chronic myeloid leukemia]. / Síndrome nefrótica por glomerulonefrite membranosa associada a leucemia mielóide crônica.

A patient developed nephrotic syndrome four years after diagnosed chronic myelogenous leukemia. Renal histology showed characteristic changes of membranous glomerulopathy. To our knowledge, this is the first reported case of membranous glomerulopathy associated with chronic myelogenous leukemia.

Glomerular hematuria in diabetics.

Three hundred and twenty outpatients with diabetes mellitus (DM) were studied to evaluate the prevalence, origin (glomerular or nonglomerular), significance and possible association of glomerular hematuria (GH) with other clinical and laboratory features. In patients with 24 h proteinuria equal or superior to 500 mg, hematuria was seen in 16 out of 22 (72.7%); for those with 24 h proteinuria between 150 and 500 mg 5 out of 23 (21.7%) had hematuria, and in the general population of diabetics studied hematuria was present in 47 patients (14.7%). It was glomerular in 43 (13.4%) patients and nonglomerular in 4 (1.3%). Red blood cell casts were observed in 15 (34.9%) out of the 43 patients with GH. Ten out of 31 patients (32.3%) with GH, for whom 24 h proteinuria was available, had negative proteinuria in a 24 h urine when analyzed by routine methods. In 18 patients with GH, clinical and laboratory findings that could suggest a second form of glomerulopathy--nondiabetic--were negative. Renal biopsy in 9 of them showed only diabetic glomerulosclerosis. We have observed a significant association between GH and the male sex (p less than 0.001), high serum creatinine levels (p = 0.0002) and 24 h proteinuria greater than 150 mg (p less than 0.001). GH was more frequent among males with DM lasting more than 10 years (p less than 0.001) and among those with retinopathy (p less than 0.001). There was no association between GH and age, type of DM, insulin requirement or hypertension.(ABSTRACT TRUNCATED AT 250 WORDS)