In vitro culture of cells exfoliated in the urine by patients with diabetes mellitus.

As an approach to facilitate the understanding of the progression of diabetic renal disease, we assessed the urine of diabetic patients and normal volunteers for the presence of cells that could be cultured in vitro. The results suggest that both normal control subjects and diabetic patients, without clinically detectable microangiopathy, exfoliate few culturable cells into the urine. In contrast, diabetics with documented retinopathy but without nephropathy exfoliate substantially higher numbers of culturable cells (5.2 cells/100 ml urine), whereas diabetics with both retinopathy and advanced nephropathy exfoliate even greater numbers of culturable cells (50.8 cells/100 ml urine). The cells that are exfoliated and culturable can be divided into five distinct cell types based on morphology at the light microscope level. The exfoliated cells proliferate at clonal density after isolation from urine and are epithelial in appearance. These data suggest that the culture of cells from urine might have diagnostic value as an early indicator of diabetic renal disease and provide a convenient, noninvasive new source of human kidney epithelial cells.

Abnormal regulation of renal kallikrein in experimental diabetes. Effects of insulin on prokallikrein synthesis and activation.

The effects of streptozotocin (STZ) diabetes and insulin on regulation of renal kallikrein were studied in the rat. 1 and 2 wk after STZ injection, diabetic rats had reduced renal levels and urinary excretion of active kallikrein. Tissue and urinary prokallikrein levels were unchanged, but the rate of renal prokallikrein synthesis relative to total protein synthesis was reduced 30-45% in diabetic rats. Treatment of diabetic rats with insulin prevented or reversed the fall in tissue level and excretion rate of active kallikrein and normalized prokallikrein synthesis rate. To further examine insulin's effects, nondiabetic rats were treated with escalating insulin doses to produce hyperinsulinemia. In these rats, renal active kallikrein increased. Although renal prokallikrein was not increased significantly by hyperinsulinemia, its synthesis was increased. As this was accompanied by proportionally increased total protein synthesis, relative kallikrein synthesis rate was not changed. Excretion of active kallikrein was unchanged, but prokallikrein excretion was markedly reduced. Therefore, increased tissue active kallikrein seen with hyperinsulinemia can be explained not only by increased synthesis but also by retention and increased activation of renal prokallikrein. These studies show that STZ diabetes produces an impairment in renal kallikrein synthesis and suggest that this disease state also impairs renal prokallikrein activation. The findings also suggest that insulin modulates renal kallikrein production, activation, and excretion.

Evidence for renal kinins as mediators of amino acid-induced hyperperfusion and hyperfiltration in the rat.

This study examined the role of tissue kallikrein and kinins in renal vasodilation produced by infusion of amino acids (AA). In rats fed a 9% protein diet for 2 wk, intravenous infusion of a 10% AA solution over 60-90 min reduced total renal vascular resistance and increased glomerular filtration rate (GFR) by 25-40% and renal plasma flow (RPF) by 23-30% from baseline. This was associated with a two- to threefold increase in urinary kinin excretion rate. Acute treatment of rats with aprotinin, a kallikrein inhibitor, resulted in deposition of immunoreactive aprotinin in kallikrein-containing connecting tubule cells and inhibited renal kallikrein activity by 90%. A protinin pretreatment abolished the rise in urinary kinins and prevented significant increases in GFR and RPF in response to AA. In a second group of rats pretreated with a B2 kinin receptor antagonist, [DArg Hyp3, Thi5,8 D Phe7]bradykinin, AA infusion raised urinary kinins identically as in untreated controls, but GFR and RPF responses were absent. Aprotinin or the kinin antagonist produced no consistent change in renal function in rats that were not infused with AA.AA-induced increases in kinins were not associated with an increase in renal kallikrein activity. Notably, tissue active kallikrein level fell 50% in AA-infused rats. These studies provide evidence that kinins generated in the kidney participate in mediating renal vasodilation during acute infusion of AA.

Skeletal muscle kallikrein. Potential role in metabolic regulation.

Skeletal muscle glucose metabolism appears to be regulated by locally derived factors as well as by systemically circulating hormones. Local factors may be particularly important during exercise, when substrate demand can increase rapidly. Numerous studies in perfused limbs suggest that the kallikrein-kinin system may participate in the regulation of substrate delivery and utilization by skeletal muscle. Evidence also suggests that kinins mediate the increase in insulin sensitivity after administration of converting enzyme inhibitors. Tissue kallikrein has been isolated and purified from rat skeletal muscles, and its level is highest in muscle with high oxidative activity. In other tissues, kallikrein synthesis is under the influence of insulin. It has not been possible to demonstrate effects of kallikrein or kinins on glucose metabolism in isolated skeletal muscle or cardiocytes. Therefore modulation of glucose metabolism by kallikrein or kinins may only be observed in intact perfused tissues or organs.

Renal kallikrein and hemodynamic abnormalities of diabetic kidney.

The relationship between renal hemodynamic abnormalities and renal kallikrein activity was studied in streptozocin-induced diabetic rats. Diabetic rats were either not treated with insulin and had plasma glucose levels greater than 400 mg/dl (severely hyperglycemic diabetic [SD]) or were treated with 1.5-1.75 U/day protamine zinc insulin and had glucose levels of 200-300 mg/dl (moderately hyperglycemic diabetic [MD]). In SD rats, kidney tissue level and excretion of active kallikrein were reduced after 3 wk compared with age-matched nondiabetic control rats (tissue, 11.7 +/- 1.9 vs. 20.5 +/- 1.8 ng/mg protein, P less than 0.005; urine, 126 +/- 12 vs. 179 +/- 10 micrograms/24 h, P less than 0.005). Despite increased kidney size, renal plasma flow (RPF) was reduced in SD rats (5.38 +/- 0.23 vs. 6.37 +/- 0.20 ml/min, P less than 0.05). Glomerular filtration rate (GFR) was not significantly lower (2.77 +/- 0.60 vs. 3.02 +/- 0.56 ml/min). In MD rats, kidney tissue level and excretion of active kallikrein were increased after 5 wk compared with age-matched nondiabetic control rats (tissue, 28.4 +/- 1.3 vs. 23.3 +/- 1.7 ng/mg protein, P less than 0.05; urine, 289 +/- 16 vs. 196 +/- 13 micrograms/24 h, P less than 0.001). In MD rats, GFR and RPF were increased (3.80 +/- 0.11 and 8.04 +/- 0.17 ml/min, respectively) compared with control rats (3.22 +/- 0.05 and 7.28 +/- 0.09 ml/min, P less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Kinin, a mediator of diabetes-induced glomerular hyperfiltration.

Renal kallikrein is increased in diabetic patients and streptozotocin (STZ)-induced diabetic rats with hyperfiltration. Chronic inhibition of renal kallikrein reduces glomerular filtration rate (GFR) and renal plasma flow (RPF) in hyperfiltering STZ-induced diabetic rats. To investigate whether these actions of kallikrein and its inhibition are kinin-mediated, we used a B2-kinin receptor antagonist (BKA). In STZ-induced diabetic rats with hyperfiltration, renal kallikrein excretion rate was significantly increased (P < or = 0.01), and kinin excretion rate was increased 57%, as compared with control rats. Left kidney GFR and RPF were measured before and during a 40-min infusion of BKA (0.5 micrograms.kg-1.min-1) or vehicle. Infusion of the kinin receptor antagonist reduced the GFR and RPF significantly. GFR was reduced by 18%, from an average baseline value of 2.07 +/- 0.11 to 1.70 +/- 0.06 ml/min, P < or = 0.001 (means +/- SE). RPF was reduced by 25%, from 6.74 +/- 0.38 to 5.06 +/- 0.17 ml/min, P < or = 0.001. Total renal vascular resistance was significantly increased during BKA infusion, P < or = 0.001. Vehicle infusion for the same period had no significant effect on GFR, RPF, or renal vascular resistance. These findings further support the hypothesis that increased renal production of kinins contributes to the renal vasodilation of diabetes.

Predicting insulin requirements for a portable insulin pump using the Biostator. Evidence for reversible insulin resistance in poorly controlled type I diabetics.

Glycemic control was achieved in 14 patients with insulin-dependent diabetes mellitus (IDDM) by 36-48-h treatment with a recently marketed clinical model, Biostator glucose controller (Life Science Instruments, Miles Laboratories, Elkhart, Indiana). Control was maintained by continuous subcutaneous insulin infusion with a portable pump, programmed using infusion profiles from the Biostator. Control of glycemic excursion with the Biostator was variable among patients. This control, reflected by the M-value or a blood glucose index (mean of pre-, peak, and 2-h postmeal levels for four meals) of each patient, correlated directly with their prior glycemic control, as assessed by hemoglobin A1c (HbA1c) level (r = 0.66, P less than 0.01 and r = 0.82, P less than 0.005, for M-value and blood glucose index, respectively). Total insulin infused by the Biostator/24 h overpredicted the subcutaneous infusion dose required on day 2 of pump treatment (183 +/- 11%, P less 0.001). Therefore, these data were not used to program the portable pump. Instead, total insulin dose was estimated using a dietary glucose/insulin (G/I) ratio. This ratio, derived from dietary total available glucose, urine glucose, and insulin dose/24 h during depot insulin treatment, accurately estimated total insulin for pump infusion (97 +/- 4%). The basal infusion rate of the Biostator between 2400 and 0600 h also exceeded the subcutaneous infusion requirement and was reduced to 40% for the initial pump basal rate. The remainder of the insulin (total minus basal) was distributed as premeal boluses according to the Biostator infusion profile for meals.(ABSTRACT TRUNCATED AT 250 WORDS)

Induction of renal kallikrein and renin gene expression by insulin and IGF-I in the diabetic rat.

The renal kallikrein-kinin system and the renin-angiotensin system are implicated in the pathogenesis of diabetic nephropathy. We have shown that renal kallikrein and renin gene expression are altered by diabetes. To investigate the cellular mechanisms responsible for these changes, we examined the effects of acute insulin and insulin-like growth factor I (IGF-I) treatment on renal kallikrein-kinin and renin-angiotensin system components. Three weeks after induction of diabetes, we measured renal kallikrein and renin mRNA levels, renal kallikrein and renal renin activity, and plasma renin activity in control and diabetic rats and diabetic rats treated with insulin or IGF-I for 2 or 5 h. In diabetic rats, kallikrein and renin mRNA levels were reduced >50% compared with control rats. Renal tissue kallikrein levels and plasma renin activity were decreased, whereas renal renin content was unchanged. Insulin increased kallikrein and renin mRNA levels after 2 h. IGF-I, at a dosage that stimulated kallikrein mRNA levels in control rats, had no effect on renal kallikrein and renin content or mRNA levels in diabetic rats. However, infusion of a fivefold higher IGF-I dosage resulted in a two- to threefold increase in kallikrein and renin mRNA levels in 2 h. These data suggest that 1) diabetes suppresses kallikrein and renin gene expression, and these abnormalities are reversed by insulin or IGF-I; and 2) the diabetic state produces resistance to IGF-I induction of kallikrein and renin gene expression. These changes in regulated synthesis of kallikrein and renin in the kidney may underlie renal vascular changes that develop in diabetes.

Urinary and renal tissue kallikrein in the streptozocin-diabetic rat.

The renal kallikrein-kinin system is thought to participate in blood pressure regulation and displays abnormalities in human hypertension, as well as in many animal models of hypertension. Urinary excretion and tissue levels of renal kallikrein were measured in streptozocin (STZ)-diabetic rats in relation to blood pressure, glycemia, and insulin treatment. In study 1, STZ-diabetic rats with marked hyperglycemia showed reduced kallikrein-like esterase excretion, compared with control rats, when first measured after 7 days of diabetes (9.9 +/- 2.5 versus 17.5 +/- 2.4 EU/24 h, P less than 0.05). This difference increased with time and, after 210 days, urinary esterase excretion in diabetic and control rats was 6.7 +/- 2.1 and 39.0 +/- 6.0 EU/24 h, respectively (P less than 0.001). Urine kallikrein, measured by radioimmunoassay, was similarly reduced in diabetic rats (40.4 +/- 8.0 versus 88.0 +/- 6.5 micrograms/24 h, at 30 days, P less than 0.001). At 120 days, systolic blood pressures were elevated in diabetic rats (P less than 0.05), and at 180 days over 60% of the diabetic rats had pressures above the highest pressures of control rats. In study 2, STZ-diabetic rats were treated with insulin for 2 wk (2 U NPH at 0800 h, or 2 U NPH at 0800 and 1600 h). In the single-dose group, with hyperglycemia similar to that of diabetic rats in study 1, kallikrein excretion was reduced as early as day 2, compared with nondiabetic rats (56.0 +/- 6.1 versus 109 +/- 9.4 micrograms/24 h, respectively, P less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Platelet function during continuous insulin infusion treatment in insulin-dependent diabetic patients.

Patients with diabetes mellitus manifest increased in vitro platelet aggregation and increased synthesis of the proaggregant and vasoconstrictor, thromboxane A2 (TXA2). We studied the effects of continuous insulin infusion treatment on platelet aggregation and arachidonic acid (AA)-stimulated platelet TXA2 synthesis (15 and 30 s post-AA, 1 mM) in 16 type I diabetic patients. Strict glycemic control was induced with the Biostator for 2 days and maintained for 12-14 days with continuous subcutaneous insulin infusion (CSII). The average premeal plasma glucose level (4/day) fell from 184 +/- 15, before treatment, to 107 +/- 6 mg/dl on the final day (P less than 0.001). After control, platelet synthesis of TXA2, measured by radioimmunoassay of its stable metabolite, immunoreactive TXB2 (iTXB2), decreased in all patients (30 s: 276 +/- 31 versus 199 +/- 28 ng iTXB2/ml/5 X 10(5) platelets; P less than 0.05). The reduction in platelet iTXB2 synthesis (15 and 30 s) was greater in poorly controlled patients (HbA1c greater than 12%; N = 8), and for all patients the decrease in iTXB2 (15 and 30 s) was correlated with the prestudy HbA1c level (15 s: r = 0.6; P less than 0.01). In contrast, platelet aggregation responses did not improve during intensive insulin treatment. The ED50 for AA (dose producing 50% maximum aggregation at 1 min) was unchanged after 2 wk of treatment and the ED50 for aggregation induced by ADP fell significantly in patients with HbA1c greater than 12% (2.8 +/- 1.3 versus 1.2 +/- 0.6 microM; P less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Native and modified LDL activate extracellular signal-regulated kinases in mesangial cells.

Glycation and/or oxidation of LDL may promote diabetic nephropathy. The mitogen-activated protein kinase (MAPK) cascade, which includes extracellular signal-regulated protein kinases (ERKs), modulates cell function. Therefore, we examined the effects of LDL on ERK phosphorylation in cultured rat mesangial cells. In cells exposed to 100 microg/ml native LDL or LDL modified by glycation, and/or mild or marked (copper-mediated) oxidation, ERK activation peaked at 5 min. Five minutes of exposure to 10-100 microg/ml native or modified LDL produced a concentration-dependent (up to sevenfold) increase in ERK activity. Also, 10 microg/ml native LDL and mildly modified LDL (glycated and/or mildly oxidized) produced significantly greater ERK activation than that induced by copper-oxidized LDL +/- glycation (P < 0.05). Pretreatment of cells with Src kinase and MAPK kinase inhibitors blocked ERK activation by 50-80% (P < 0.05). Native and mildly modified LDL, which are recognized by the native LDL receptor, induced a transient spike of intracellular calcium. Copper-oxidized (+/- glycation) LDL, recognized by the scavenger receptor, induced a sustained rise in intracellular calcium. The intracellular calcium chelator (EGTA/AM) further increased ERK activation by native and mildly modified LDL (P < 0.05). These findings demonstrate that native and modified LDL activate ERKs 1 and 2, an early mitogenic signal, in mesangial cells and provide evidence for a potential link between modified LDL and the development of glomerular injury in diabetes.

Thyrotoxicosis without elevated serum triiodothyronine levels during diabetic ketoacidosis.

Thyrotoxicosis with a normal serum triiodothyronine (T3) concentration has been described with a variety of acute and chronic illnesses occurring in association with thyrotoxicosis. We describe the first case to our knowledge of thyroxine (T4) toxicosis in a 16-year-old boy with diabetic ketoacidosis. Although the clinical manifestations of hyperthyroidism were mild, thyromegaly and persistent tachycardia suggested thyrotoxicosis. Serum T4 levels were elevated; however, the serum T3 level was normal. Measurement of reverse T3 (rT3) initially revealed an elevated level that decreased over several days of T3 levels increased into the toxic range. Peripheral conversion of T4 to T3 was apparently inhibited by diabetic ketoacidosis and there was a concomitant increase in rT3 levels, suggesting that conversion of T4 to rT3 was increased during acute ketoacidosis. Assessment of thyroid function based on serum T3 levels in diabetics may be misleading during ketoacidosis or uncontrolled diabetes.

Role of kinins in the renal response to enalaprilat in normotensive and hypertensive rats.

This study examined the role of endogenous kinins in the alteration of renal hemodynamics induced by low-dose converting enzyme inhibition in hydropenic normotensive rats and in the nonclipped kidney of hydropenic two-kidney, one clip hypertensive rats. Infusion of a bradykinin B2 receptor antagonist (D-Arg0,[Hyp3,Thi5,8,D-Phe7]-bradykinin, 1 or 10 micrograms.kg-1.min-1) did not alter renal function of normotensive rats. In a second series of experiments, infusion of enalaprilat at 0.1 mg.kg-1.h-1 increased renal blood flow (P < .01) and decreased renal vascular resistance (P < .01). The superimposition of the kinin antagonist at 1 micrograms.kg.min-1 during the enalaprilat infusion decreased renal blood flow to a value similar to the preenalaprilat baseline and significantly different from the mean of the two enalaprilat periods before and after the addition of the kinin antagonist--the "mean effect of enalaprilat." The decrease in renal blood flow induced by the kinin antagonist was associated with an increase in renal vascular resistance above the mean effect of enalaprilat (P < .025). In two-kidney, one clip hypertensive rats, systemic infusion of enalaprilat augmented the hemodynamics of the nonclipped kidney by a degree similar to that in normotensive rats. In contrast to normotensive rats, superimposition of the kinin antagonist did not alter the enalaprilat-induced change in blood flow or vascular resistance of the nonclipped kidney. The results of this study suggest that endogenous kinins contribute to the increased renal function induced by low-dose converting enzyme inhibition in hydropenic normotensive rats but appear to contribute less to the enalaprilat-induced alterations of renal function in the nonclipped kidney of two-kidney, one clip hypertensive rats.

Urinary kallikrein excretion in insulin-dependent diabetes mellitus and its relationship to glycemic control.

The renal kallikrein-kinin system is thought to be involved in vasoregulatory and epithelial ion-transporting processes. Renal kallikrein has not been studied in patients with diabetes mellitus, a disease in which abnormalities of renal hemodynamics and electrolyte handling occur. The urinary excretion of this kallikrein was measured in 20 type I diabetic patients and 10 normal subjects. On a 120-meq Na diet, daily kallikrein excretion, determined by both esterase activity and direct RIA, in 12 poorly controlled diabetic patients [hemoglobin A1c (HbA1c) = 14.2 +/- 0.5% (mean +/- SEM)] was significantly greater (P less than 0.05) than excretion in 8 diabetic patients in good to moderately good control (HbA1c = 9.4 +/- 0.5%) or in 10 normal subjects. In these groups, urinary esterase activities were 9.4 +/- 1.0, 6.1 +/- 1.4, and 6.7 +/- 0.5 esterase units/24 h, respectively. Corresponding excretion values of immunoreactive kallikrein were 171 +/- 14, 118 +/- 26, and 123 +/- 11 micrograms/24 h. Creatinine clearances were similar in the three groups. Urinary kallikrein was also measured in 8 diabetic and 8 normal subjects during 7 subsequent days of 10 meq Na intake. It increased less in diabetic patients than in normal subjects during Na depletion (P less than 0.02). The increase in urinary kallikrein in the diabetic patients was inversely related to their HbA1c levels (r = 0.88; P less than 0.01). The effect of glycemic control on urinary kallikrein excretion was determined in nine diabetic patients. Initial glycemic control was achieved using an artificial endocrine pancreas (Biostator) and was maintained by continuous sc insulin infusion with a portable pump. Before glycemic control, urinary kallikrein was 190 +/- 30 micrograms/24 h (by RIA). After 8-12 days of glycemic control, excretion fell to 144 +/- 23 micrograms/24 h (P less than 0.02). The abnormalities in kallikrein excretion in diabetic patients were not correlated with differences in water, electrolyte, protein, glucose, or aldosterone excretion in any of the studies. These results show that kallikrein excretion was increased in patients with poorly controlled insulin-dependent diabetes, and excretion rose less in diabetic subjects with low Na intake than in normal subjects. Strict glycemic control decreased urinary kallikrein excretion. These findings suggest that the renal kallikrein-kinin system is functioning abnormally in diabetes mellitus.

Failure to visualize adrenal glands in a patient with bilateral adrenal hyperplasia.

A patient with clinical and biochemical evidence of Cushing's disease and severe hyperlipidemia underwent an adrenal imaging procedure with NP-59 (6 beta-[131I]iodomethyl-19-norcholesterol), without visualization of either gland. Correction of the hyperlipidemia followed by repeated adrenal imaging resulted in bilateral visualization. A pituitary tumor was removed at surgery, confirming the diagnosis of Cushing's disease.

Primary hyperparathyroidism in a twin pregnancy and review of fetal/maternal calcium homeostasis.

BACKGROUND: Hyperparathyroidism occurs rarely in pregnancy; this is the first reported case in a twin gestation. Management of this unusual case is described and an overview of fetal/maternal calcium homeostasis is discussed. METHODS: The patient presented at 33 weeks' gestation with hypertension and premature labor. Serum calcium and phosphorus were 14.6 and 1.7 mg/dL, respectively. An intact parathyroid hormone (PTH) level was 243 pg/mL (normal, 10-65). RESULTS: The patient was treated with parenteral saline hydration and oral phosphate supplementation that was continued through week 37. Although the calcium remained elevated between 12.6 and 13.3 mg/dL, medical therapy was continued because of the risks of surgery in the third trimester. Alternative medical treatments (bisphosphonates, calcitonin) were considered ill advised in pregnancy. The patient remained asymptomatic without further labor, and at week 37, fraternal twins were delivered by cesarean section. The infants were monitored closely and experienced no hypocalcemic symptoms after delivery. Postpartum, the mother's parathyroid scan and ultrasound were negative. She underwent neck exploration and a single 700-mg adenoma was removed. Transient asymptomatic hypocalcemia (7.5 mg/dL) occurred postoperatively, and she was placed on oral calcium (1500 mg/day) and calcitriol (0.25 mg/day). These were stopped at 8 weeks, when both PTH and parathyroid hormone-related peptide levels were normal. CONCLUSION: Mother and infants continue to do well after 18 months. This case provides an interesting setting to consider the interrelationships between elevated maternal PTH and the fetal/placental factors that regulate calcium metabolism in pregnancy.

Pituitary adenocarcinoma in an acromegalic patient: response to bromocriptine and pituitary testing: a review of the literature on 36 cases of pituitary carcinoma.

There are 36 reported cases of metastatic pituitary carcinoma and almost half (44%) of these were associated with syndromes of hormonal hypersecretion. The case of a 56-year-old acromegalic man with cervical lymphatic and spinal metastases from a primary pituitary carcinoma is described. Elevated basal levels of plasma growth hormone (GH) and insulin growth factor-1/Somatomedin C (IGF-1/SmC) were found. GH levels did not increase after TRH or LHRH administration but decreased after L-Dopa and glucose. Immunostaining of the metastatic tumor for GH and electron microscopy findings confirmed the diagnosis of pituitary GH-secreting carcinoma. Striking clinical improvement and a 46% decrease in plasma GH levels were observed with bromocriptine treatment, although IGF-1/SmC levels increased during therapy. The clinical course of most reported cases of pituitary adenocarcinoma has been one of progressive intracranial expansion of a pituitary neoplasm. In only 25% were metastatic lesions discovered antemortem, and disabling symptomatology caused by metastases was rare. Only four previously reported patients of 36 with pituitary carcinoma had acromegaly.