Miroslaw Smogorzewski

Intensive blood-pressure control in hypertensive chronic kidney disease.

BACKGROUND In observational studies, the relationship between blood pressure and end-stage renal disease (ESRD) is direct and progressive. The burden of hypertension-related chronic kidney disease and ESRD is especially high among black patients. Yet few trials have tested whether intensive blood-pressure control retards the progression of chronic kidney disease among black patients. METHODS We randomly assigned 1094 black patients with hypertensive chronic kidney disease to receive either intensive or standard blood-pressure control. After completing the trial phase, patients were invited to enroll in a cohort phase in which the blood-pressure target was less than 130/80 mm Hg. The primary clinical outcome in the cohort phase was the progression of chronic kidney disease, which was defined as a doubling of the serum creatinine level, a diagnosis of ESRD, or death. Follow-up ranged from 8.8 to 12.2 years. RESULTS During the trial phase, the mean blood pressure was 130/78 mm Hg in the intensive-control group and 141/86 mm Hg in the standard-control group. During the cohort phase, corresponding mean blood pressures were 131/78 mm Hg and 134/78 mm Hg. In both phases, there was no significant between-group difference in the risk of the primary outcome (hazard ratio in the intensive-control group, 0.91; P=0.27). However, the effects differed according to the baseline level of proteinuria (P=0.02 for interaction), with a potential benefit in patients with a protein-to-creatinine ratio of more than 0.22 (hazard ratio, 0.73; P=0.01). CONCLUSIONS In overall analyses, intensive blood-pressure control had no effect on kidney disease progression. However, there may be differential effects of intensive blood-pressure control in patients with and those without baseline proteinuria. (Funded by the National Institute of Diabetes and Digestive and Kidney Diseases, the National Center on Minority Health and Health Disparities, and others.)

Polymorphonuclear Leukocytes in Non-Insulin-dependent Diabetes Mellitus: Abnormalities in Metabolism and Function

A large body of evidence indicates that the cytosolic calcium ([Ca2+]i) level is elevated in many cells in both patients with insulin-dependent diabetes mellitus (IDDM) and those with noninsulin-dependent diabetes mellitus (NIDDM) [1-6]. This phenomenon led Levy and Gavin [7] to propose that many of the complications of diabetes mellitus are, at least in part, related to an elevation in [Ca2+]i levels. This is plausible because other conditions in which [Ca2+]i levels are elevated, such as chronic renal failure [8] and phosphate depletion [9-12], are associated with organ dysfunctions similar to those seen with diabetes mellitus. However, a relation between the elevation in [Ca2+]i levels and a specific cell dysfunction in diabetes mellitus has not yet been documented. The mechanisms responsible for the elevation in [Ca2+]i levels in patients with diabetes mellitus are not known. Hyperglycemia may be associated with increased calcium influx into cells and a corresponding increase in [Ca2+]i levels [13]. However, data on the relation among the degree of hyperglycemia, the magnitude of the increase in [Ca2+]i, and cell dysfunction are not available. Furthermore, it is not known whether the reversal of hyperglycemia by insulin therapy or by the use of oral hypoglycemic agents is followed by the return of [Ca2+]i levels and cell function to normal. Phagocytosis by polymorphonuclear leukocytes (PMNLs) is impaired in patients with IDDM and NIDDM [14, 15]. However, data on [Ca2+]i levels in PMNLs and on the relations among hyperglycemia, [Ca2+]i levels in PMNLs, and phagocytosis are not available. The study of these accessible cells in patients with diabetes mellitus may provide an opportunity to explore these issues. We therefore examined PMNLs from patients newly diagnosed with NIDDM in order to delineate the interactions among hyperglycemia, [Ca2+]i levels, and phagocytosis. Methods We studied 22 controls and 34 patients with NIDDM to evaluate [Ca2+]i levels and phagocytosis in PMNLs. At the time of diagnosis, we obtained blood samples for the study of PMNL metabolism and function. All patients received the oral hyperglycemic agent glyburide (5 to 20 mg/d; mean dose, 7 2.8 mg/d). Only 15 patients returned for follow-up after 3 months of therapy with this drug; no specific reasons were given for why the remaining 19 patients did not return for follow-up. All biochemical variables were assessed in the patients but not in the controls. The normal values for our laboratory are provided. Separation of Polymorphonuclear Leukocytes Peripheral venous blood was drawn under sterile conditions into vacutainers containing 20 U of preservative-free heparin (Gibco Laboratories, Grand Island, New York) per 1.0 mL of blood. Polymorphonuclear leukocytes were isolated from the whole blood according to the method described by Ferrante and Thong [16]. Fresh heparinized blood was layered in 3.5-mL aliquots over Ficoll-Hypaque solution with a density of 1.114 g/mL (Mono-Poly Resolving Medium, Flow Laboratories, McLean, Virginia) and centrifuged at 300 g for 35 min at room temperature. This procedure resulted in the separation of mononuclear and polymorphonuclear cells into two distinct bands, with the red blood cell pellet at the bottom of the tube. The PMNL layer was aspirated with a Pasteur pipette and washed twice in Hanks balanced salt solution. The purity of cells as determined by Wright stain was greater than 97%, and the viability of cells exceeded 98% as assayed by the trypan blue exclusion method. Determination of Phagocytosis We estimated the rate of ingestion by PMNLs spectrophotometrically according to the bioassay described by Southwick and Stossel [17]. Briefly, PMNLs were fed oil droplets containing oil red O and coated with Escherichia coli lipopolysaccharide (2 mg per experiment). These particles were previously opsonized in fresh autologous serum. After the PMNLs and the particles were incubated for 5 min at 37 C, the reaction was rapidly stopped by adding ice-cold isotonic saline that contained 1 mmol/L N-ethylmaleimide, which poisons the cells and stops ingestion. Uningested particles were separated from cells that contained ingested particles by centrifugation at 250 g for 10 min. Oil red O was extracted from the cell pellet with dioxane, and the optical density of the extract was measured using a Perkin Elmer Lambda 2 UV/VIS spectrophotometer (Perkin Elmer, Norwalk, Connecticut) at a wavelength of 525 nm. The ingestion rate was expressed in micrograms of oil engulfed per 107PMNLs per minute. We purchased all reagents from Aldrich Chemical (Milwaukee, Wisconsin). Lipopolysaccharide E. coli 026:B6 was obtained from Boivin preparation (Difco Laboratories, Detroit, Michigan). Assay of Adenosine Triphosphate in Polymorphonuclear Leukocytes We measured the ATP content of PMNLs according to the method described by Lundin and colleagues [18]. We added an aliquot of 0.5 mL of 0.6 M ice-cold perchloric acid to 5 106PMNLs in 0.5 mL of RPMI (Gibco Laboratories). After 10 minutes of extraction on ice, we added 62.5 L of 2 M potassium carbonate, and the mixture was centrifuged at 10 500 g for 10 min. The supernatant was removed and immediately frozen with liquid nitrogen and stored at 70 C. On the day of the assay, we added 50 L of the supernatant to 950 L of 40 mmol/L Tris buffer (pH, 7.4) and diluted this mixture 20 times with distilled water. Samples of 50 L were assayed for ATP by the firefly luciferase assay with LAD 535 luminometer (Turner Design, Sunnyvale, California). Adenosine triphosphate standards were prepared with 40 mmol/L Tris buffer, distilled water, and amounts of RPMI, perchloric acid, and potassium carbonate similar to those in the PMNL preparation. Measurements of Cytosolic Calcium We measured resting levels of [Ca2+]i in PMNLs with Fura 2-AM (Sigma, St. Louis, Missouri). A sample of 5 106PMNLs was washed with solution 1, which contained 132 mmol/L sodium chloride; 3 mmol/L potassium chloride; 1 mmol/L magnesium sulfate; 1.2 mmol/L monosodium acid phosphate; 10 mmol/L D-glucose; 10 mmol/L Hepes; and 0.02 mmol/L calcium chloride (pH was adjusted to 7.4 with Tris buffer) and was then spun at 300 g for 15 min. The pellet was resuspended in 490 L of this solution and 10 L of Fura 2-AM dissolved in dimethyl sulfoxide, giving a final concentration of 4 mol/L of Fura 2. The mixture was then incubated in a water bath of 37 C for 30 minutes. After this incubation, the cells were washed and resuspended in solution 1. Cytosolic calcium levels were measured with Perkin Elmer fluorescence spectrophotometer (model LS 513) at excitation wavelengths of 340 and 380 nm and an emission wavelength of 510 nm, with slits of 10 mm and 20 mm, respectively. We added a 100- L aliquot of the PMNL suspension to a spectrophotometer cuvette that contained 1.9 mL of solution 2, which was the same as solution 1 except that its calcium chloride concentration was 1 mmol/L. Autofluorescence from cells or added reagents (or both) was monitored during each experiment and was not found to be a significant factor. We evaluated maximal fluorescence and minimal fluorescence with 0.05% Triton and 5 mmol/L ethylene glycol tetra-acetic acid in Tris base buffer (pH, 8.0), respectively. Cytosolic calcium was calculated using the Grynkiewicz equation [19], and the dissociation constant for Ca2+-Fura 2 was assumed to be 225 nmol/L. Biochemical Measurements The concentration of calcium in serum was determined by Perkin Elmer spectrophotometer model 505 of phosphorus; that of creatinine by an autoanalyzer (Technicon, Tarrytown, New York); that of serum glucose by a multichannel analyzer (Beckman, Irvine, California); and that of hemoglobin A1c (HbA1c) by an ion exchange chromatography according to a modification of the method of Trivelli and colleagues [20]. Serum parathyroid hormone (PTH) levels were estimated with radioisotopic assay (Intact PTH-Nichols Institute Diagnostics). The normal value of PTH is 10 to 65 pg/mL. Statistical analysis was done using a paired and an unpaired t-test, and the relation between two variables was determined by the correlation coefficient. Data are reported as mean SD unless otherwise specified. Results The study included 22 controls and 34 patients with NIDDM. The patients were examined at the time of their visit to the outpatient clinic and consisted of 20 women and 14 men (mean age, 45 12.8 years; range, 30 to 71 years). No patients had infection, nor were they receiving any medications. The diagnosis of NIDDM was made by measurements of fasting hyperglycemia in the absence of ketonemia [21]. However, some of these patients may have had late-onset IDDM. All patients had normal blood pressure (mean, 127 19.8/77 11.1 mm Hg). The mean body mass index was 32.6 9.3 kg/m2 for women and 30.0 6.4 kg/m2 for men. The body mass index was normal in 2 women, in the overweight range in 4, and in the obese range in 11. It was normal in 2 men, in the over-weight range in 7, and in the obese range in 8. Table 1 provides the clinical and biochemical data of the patients at the time of diagnosis. They had hyperglycemia, elevated blood levels of HbA1c, normal blood levels of creatinine and PTH, and blood cholesterol levels in the upper normal range. Ten patients had elevated blood levels of triglycerides, 19 patients had elevated low-density lipoprotein cholesterol levels, and 16 patients had decreased high-density lipoprotein cholesterol levels. The controls were 9 women and 13 men (mean age, 35 4.9 years; range, 26 to 45 years). Table 1. Biochemical Profile of Patients with Non-Insulin-Dependent Diabetes Mellitus* In the patients with NIDDM, the basal [Ca2+]i levels in PMNLs (68 9.6 nmol/L) were significantly higher than those in the controls (43 4.9 nmol/L) (P < 0.01) (Figure 1). We observed a direct and significant correlation between [Ca2+]i levels in PMNLs and fasting serum glucose levels (r = 0.80; P < 0.01) (Figure 2). The PMNLs f

Impaired Phagocytosis in Dialysis Patients: Studies on Mechanisms

Dialysis patients have increased susceptibility to infection and this is, in part, due to impaired phagocytic and bactericidal activities of polymorphonuclear leukocytes (PMNL). The mechanisms responsible for the reduced phagocytosis are not known. Dialysis patients have elevated blood levels of parathyroid hormone (PTH), and available data indicate that PMNL is a target cell for PTH. Chronic exposure to excess PTH may cause accumulation of calcium in PMNL which in turn could adversely affect cellular events leading to their dysfunction. We studied phagocytosis, resting levels of cytosolic calcium [( Ca2+]i), ATP content and the rise in [Ca2+]i in response to ligation of Fc gamma RIII receptors with 3G8 monoclonal antibody in PMNL from 37 dialysis patients and 48 normal subjects. The PMNL from the dialysis patients displayed impaired phagocytosis, elevated resting levels of [Ca2+]i, decreased ATP content and a smaller rise in [Ca2+]i in response to various doses of 3G8 monoclonal antibody as compared to values obtained in PMNL of normal subjects. Our results suggest that derangements in cellular metabolism and possibly an abnormality in Fc gamma RIII interaction with antibody and/or the consequences of such interaction are responsible, at least in part, for the impaired phagocytosis of PMNL of dialysis patients. Our data are consistent with the notion that excess PTH may play an important role in the processes leading to impaired phagocytosis.

Abnormalities in hepatic lipase in chronic renal failure: role of excess parathyroid hormone.

Post-heparin hepatic lipase activity is reduced in chronic renal failure (CRF). This could be due to reduced synthesis, decreased activity, and/or impaired secretion of the enzyme. Further, the factor(s) responsible for such derangements are not elucidated. We examined hepatic lipase metabolism in normal, 6-wk-old CRF rats, CRF-PTX (parathyroidectomized) rats, and CRF and normal rats treated with verapamil (CRF-V, normal-V) using liver homogenate, hepatic cell culture for 8 h, and in vitro liver perfusion. The Vmax of hepatic lipase in liver homogenate was significantly (P < 0.01) reduced and the Km was significantly (P < 0.01) increased in CRF rats, but the values were normal in CRF-PTX, CRF-V, and normal-V rats. Culture of hepatic cells for 8 h was associated with an increase in hepatic lipase activity but the increment in CRF rats was significantly (P < 0.01) lower than that of normal, CRF-PTX, CRF-V, and normal-V rats. Both parathyroid hormone (PTH)-(1-84) and 1-34 inhibited the production of hepatic lipase in cultured cells from normal, CRF-PTX, CRF-V, and normal-V rats. The expression of the mRNA of the hepatic lipase was significantly reduced in CRF animals with the ratio between it and that of house keeping gene G3DPH being 15 +/-3% compared to 40 +/- 1.3% in normal, 44+/-2.9% CRF-PTX, 44 +/- 5.4% in CRF-V, and 39 +/- 3.9% in normal-V rats. Infusion of heparin to the in vitro hepatic perfusion system increased the activity of hepatic lipase in the effluent in all groups of rat except in CRF animals. Infusion of PTH-(1-34) in dose of 10(-6) M into the liver perfusion system inhibited the increase in post-heparin hepatic lipase activity. The data show that in CRF (a) the mRNA of hepatic lipase is downregulated, and hepatic lipase production, activity and release are impaired, (b) that this is due to the state of secondary hyperparathyroidism of CRF since both acute and chronic excess of PTH were associated with these abnormalities, (c) and that prevention of excess PTH by PTX of CRF rats or blocking the effect of PTH by treatment with verapamil corrected the derangement in hepatic lipase metabolism.

Parathyroid hormone-parathyroid hormone related protein receptor messenger RNA is present in many tissues besides the kidney.

Parathyroid hormone (PTH) acts on a large number of cells derived from many different tissues that are not traditional targets (kidney and bone) for their action. Also, the acute exposure of many of these cells to PTH resulted in the generation of cAMP. These observations are consistent with the presence of PTH receptors on these cells. However, there is no evidence that the cells that are not traditional targets for PTH, express the receptor of the hormone. The cloning of the PTH-PTH related protein (PTH-PTHrP) receptor provided the tool to examine whether these cells contain the mRNA for this receptor. Poly A+RNA from a variety of rat tissues was probed with a 1,200-bp fragment of the cDNA of the PTH-PTHrP receptor by the Northern blot technique. We found that mRNA for the PTH-PTHrP receptor is present in the heart, brain, spleen, lung, liver, skeletal muscle, kidney and testis. Transcripts of 2.4 kb were found in all these tissues with the strongest expression in the kidney. In addition, smaller RNAs were detected in the kidney (approximately 1.8 kb) and testis (1.5 kb). These results indicate that many cell types express the PTH-PTHrP receptor gene. The data provide a possible explanation for the direct effects of PTH on so many cells and for the understanding of the harmful effects of chronic excess of PTH on the function of many organs in chronic renal failure.

Relationship between Ambulatory BP and Clinical Outcomes in Patients with Hypertensive CKD

BACKGROUND AND OBJECTIVES Abnormal ambulatory BP (ABP) profiles are commonplace in CKD, yet the prognostic value of ABP for renal and cardiovascular outcomes is uncertain. This study assessed the relationship of baseline ABP profiles with CKD progression and subsequent cardiovascular outcomes to determine the prognostic value of ABP beyond that of clinic BP measurements. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS Between 2002 and 2003, 617 African Americans with hypertensive CKD treated to a clinic BP goal of <130/80 mmHg were enrolled in this prospective, observational study. Participants were followed for a median of 5 years. Primary renal outcome was a composite of doubling of serum creatinine, ESRD, or death. The primary cardiovascular outcome was a composite of myocardial infarction, hospitalized congestive heart failure, stroke, revascularization procedures, cardiovascular death, and ESRD. RESULTS Multivariable Cox proportional hazard analysis showed that higher 24-hour systolic BP (SBP), daytime, night-time, and clinic SBP were each associated with subsequent renal (hazard ratio, 1.17-1.28; P<0.001) and cardiovascular outcomes (hazard ratio, 1.22-1.32; P<0.001). After controlling for clinic SBP, ABP measures were predictive of renal outcomes in participants with clinic SBP <130 mmHg (P<0.05 for interaction). ABP predicted cardiovascular outcomes with no interaction based on clinic BP control. CONCLUSIONS ABP provides additional information beyond that of multiple clinic BP measures in predicting renal and cardiovascular outcomes in African Americans with hypertensive CKD. The primary utility of ABP in these CKD patients was to identify high-risk individuals among those patients with controlled clinic BP.

Hypokalemic effects of intravenous infusion or nebulization of salbutamol in patients with chronic renal failure : comparative study

To examine and compare the efficacy and safety of different routes of administration of salbutamol in treating hyperkalemia, 15 patients with chronic renal failure (blood urea nitrogen > 80 mg/dL, serum creatinine > 8.0 mg/dL) were enrolled to sequentially receive either intravenous infusion (0.5 mg) or nebulization (10 mg) of salbutamol. Five of these patients (33.3%) did not respond to the intravenous salbutamol and were excluded from the study. Both treatments significantly decreased plasma potassium in 10 patients and the decrease was sustained for at least 3 hours. After infusion, the maximal reduction in plasma potassium levels was 0.92 +/- 0.10 mEq/L and occurred after 30 minutes. On the other hand, the maximal reduction in plasma potassium after nebulization (0.85 +/- 0.13 mEq/L) was similar to that after infusion, but it occurred after 90 minutes. Insulin and blood glucose increased, whereas blood pH, PCO2, sodium, osmolality, and blood pressure did not change after either treatment. Heart rate increased significantly after both treatments, but less after nebulization than after infusion. It is concluded that both infusion and nebulization are simple, effective, and safe therapeutic modalities for the treatment of hyperkalemia in patients with chronic renal failure. Infusion should be used in patients requiring a rapid decrease in plasma potassium; nebulization, on the other hand, should be used in patients with coronary artery diseases.

Elevated cytosolic calcium and impaired proliferation of B lymphocytes in type II diabetes mellitus.

Patients with diabetes mellitus have increased susceptibility to infection attributable, at least in part, to defective function of polymorphonuclear leukocytes (PMNLs) and B cells. Certain data suggest that cytosolic calcium ([Ca2+]i) is elevated in various cells in diabetes, and high [Ca2+]i adversely affects cell function. Indeed, the [Ca2+]i of PMNLs of diabetic patients is elevated, and phagocytosis of the PMNLs is impaired. The current study examines whether the [Ca2+]i of B cells is also elevated in diabetes and whether this derangement impairs B cell function. We studied 32 patients with non-insulin-dependent diabetes mellitus (NIDDM) and eight normal subjects. All patients had hyperglycemia (11.6 +/- 0.80 mmol/L) and elevated HbA1c (13.2% +/- 0.99%). The basal levels of [Ca2+]i of the B cells (113 +/- 3.3 nmol/L) were significantly (P < 0.01) higher than the values in normal subjects (85 +/- 1.7 nmol/L). There was a direct and significant correlation (r = 0.88; P < 0.01) between the [Ca2+]i of B cells and the blood levels of glucose. Proliferation of B cells in response to Staphylococcus aureus Cowan I (SAC) was significantly impaired in these patients (7.3 +/- 0.48 x 10(3) cpm v 12.5 +/- 0.61 x 10(3) cpm in normal subjects). Normalization of blood glucose with the hypoglycemic agents, glyburide, was associated with the return of both [Ca2+]i of B cells and their proliferation in response to SAC to normal. The results show that hyperglycemia in type II diabetes mellitus is associated with a significant increase in [Ca2+]i of B cells and with a decrease in their proliferation in response to mitogen. These derangements are reversed after the correction of the hyperglycemia. The data of the current study and those previously reported in PMNLs provide for a new pathogenetic process underlying the dysfunction of these cells in diabetes mellitus.

Verapamil prevents chronic renal failure-induced abnormalities in lipid metabolism.

Hypertriglyceridemia is common in chronic renal failure (CRF); this derangement is due to decreased peripheral removal of triglycerides. Certain data indicate that the state of secondary hyperparathyroidism of CRF is, at least in part, responsible for derangements in lipid metabolism. It has been proposed that chronic excess of parathyroid hormone exerts its deleterious effects on many organs through its ability to raise basal levels of cytosolic calcium. Prevention of the latter by a calcium channel blocker is followed by the correction of organ dysfunctions. The present study examined the effect of treatment of CRF rats with verapamil on several parameters of lipid metabolism. Chronic renal failure rats displayed hypertriglyceridemia, fat intolerance, reduced postheparin plasma lipoprotein and hepatic lipase activities, decreased hepatic lipase in liver homogenate, and elevated calcium content in liver and epididymal fat. Treatment of the CRF rats with verapamil prevented all these derangements in lipid metabolism. These effects of verapamil were similar to those produced by parathyroidectomy of CRF rats. The data are consistent with the formulation that chronic excess of parathyroid hormone increases the calcium burden of liver and adipose tissue and consequently impairs the synthesis and/or release of lipoprotein and hepatic lipases. Reduced availability of these enzymes in plasma results in impared peripheral removal of triglycerides, leading to hypertriglyceridemia.

Relationship of Left Ventricular Hypertrophy and Diastolic Function With Cardiovascular and Renal Outcomes in African Americans With Hypertensive Chronic Kidney Disease

African Americans with hypertension are at high risk for adverse outcomes from cardiovascular and renal disease. Patients with stage 3 or greater chronic kidney disease have a high prevalence of left ventricular (LV) hypertrophy and diastolic dysfunction. Our goal was to study prospectively the relationships of LV mass and diastolic function with subsequent cardiovascular and renal outcomes in the African American Study of Kidney Disease and Hypertension cohort study. Of 691 patients enrolled in the cohort, 578 had interpretable echocardiograms and complete relevant clinical data. Exposures were LV hypertrophy and diastolic parameters. Outcomes were cardiovascular events requiring hospitalization or causing death; a renal composite outcome of doubling of serum creatinine or end-stage renal disease (censoring death); and heart failure. We found strong independent relationships between LV hypertrophy and subsequent cardiovascular (hazard ratio, 1.16; 95% confidence interval, 1.05–1.27) events, but not renal outcomes. After adjustment for LV mass and clinical variables, lower systolic tissue Doppler velocities and diastolic parameters reflecting a less compliant LV (shorter deceleration time and abnormal E/A ratio) were significantly (P<0.05) associated with future heart failure events. This is the first study to show a strong relationship among LV hypertrophy, diastolic parameters, and adverse cardiac outcomes in African Americans with hypertension and chronic kidney disease. These echocardiographic risk factors may help identify high-risk patients with chronic kidney disease for aggressive therapeutic intervention.