New Concepts in Diabetes and Its Treatment - part 5 pdf

Potassium administration is often required. This cation may be normal
in serum even if the total body content is decreased. When the plasma level
is low, 30–40 mmol/h of potassium should be infused, and a lower dose
(20 mmol/h) should also be given when serum potassium is normal, because
with the beginning of therapy a further fall in serum potassium occurs as a
result of the effect of insulin (which causes a shift of potassium into the cells)
and fluid replacement (that dilutes serum potassium). ECG represents a useful
tool to assess intracellular potassium concentration, showing flat or inverted
T waves when intracellular potassium is low and peaked T waves when in-
tracellular potassium is high.
Bicarbonate administration isonly required inpatients with severe acidosis
(pH=7). Bicarbonate should be given at a slow rate (about 44 mEq during 1
or 2 h) and discontinued when the pH rises to 7.1. In DKA, 2,3-diphospho-
glycerate (2,3-DPG) is low in red cells, which decreases oxygen delivery. This
is counterbalanced by acidosis, which favors oxygen delivery. A rapid correc-
tion of acidosis with bicarbonate may leave the effect of the 2,3-DPG unop-
posed, causing impaired oxygen release which, in presence of volume depletion
and reduced tissue perfusion may favor the development of tissue hypoxia
and lactic acidosis.
In presence of infections, antibiotic therapy should be employed. When
the patient is comatose, insert a nasogastric tube, use a urinary catheter (if
no urine passes within 3 h) and heparinize in case of hyperosmolar coma
development or in presence of thrombosis risk factors.
After the recovery from a diabetic ketoacidotic episode, it is useful to
accurately review the causes to reduce the risk of recurrence.
Hyperosmolar Nonketotic Syndrome
Hyperosmolar nonketotic syndrome (HNKS) is an acute complication
observed most often in type 2 diabetic patients and is characterized by symp-
toms and signs due to volume depletion (caused by excessive hyperglycemia
and consequent hyperosmolality and osmotic diuresis), with varying degree
of clouding of sensorium, ranging from absence of mental impairment (about

10%) to frank coma (about 10%). HNKS is a serious complication, which
entails a mortality rate as high as ?40%.
Pneumonia (favored by sensory clouding which facilitates aspiration of
oropharyngeal secretions) may develop in HNKS patients, as well as other
infections. The dehydration elevates plasma viscosity and may favor throm-
bosis. Disseminated intravascular coagulation (DIC) may also occur, with
bleeding manifestations.
108Belfiore/Iannello
Laboratory findings include a marked hyperglycemia (usually higher than
that occurring in DKA, reaching a level of ?800 mg/dl or 44 mmol/l) which
causes increase in serum osmolality (which may be as high as ?350 mosm/l),
whereas sodium is normal or slightly changed. Urea nitrogen and creatinine
are elevated, together with inorganic acids (phosphates and sulfates) because
of prerenal azotemia consequent to volume depletion. In contrast to DKA,
in HNKS the metabolic acidosis is absent or mild, and bicarbonates are slightly
changed. When present, acidosis is due to retention of inorganic acids (see
above), i.e. a small amount of ketone bodies as well as a certain amount of
lactate (due to tissue hypoperfusion consequent to volume depletion).
The extreme hyperglycemia with the ensuing hyperosmolality may be
favored by the abundant hyperglycemic diuresis in patients who are unable to
compensate the large fluid loss with urine by adequate water drinking, as it
often occurs in old patients, who have an attenuated sensation of thirst and
who often live alone or in nursing homes. However, it should be kept in
mind that HNKS may be precipitated by several factors, including infections,
cerebrovascular events, hypertonic peritoneal dialysis, parenteral nutrition or
administration of the osmotic agent mannitol or diuretics as well as corticoste-
roids and phenytoin.
The lack of acidosis in HNKS may be the result of several factors.
(1) HNKS develops in type 2 diabetic patients, who possess a varying degree
of residual endogenous insulin secretion. Since lipolysis is more sensitive to

insulin than the glucose homeostatic mechanisms, itis possible that the residual
insulin secretion, while unable to stimulate glucose utilizaton and to repress
hepatic glucose production, is able to refrain lipolysis, thus limiting the FFA
afflux to liver and therefore the ketogenic process. (2) The endogenously se-
creted insulin reaches, through the portal vein, the liver, which is insulinized
to a sufficient degree to prevent activation of ketogenesis (i.e. to allow glucose
to be utilized in sufficient amount to produce enough malonyl-CoA, which
inhibits the ketogenic process at the level of CPT-1. (3) There may be glucagon
resistance, which prevents glucagon to exert its ketogenic effects (see under
DKA). (4) There may be an enhanced activity of the Cori cycle, with increased
afflux of lactate to the liver, where it may be in part metabolized to malonyl-
CoA, thus refraining ketogenesis.
HNKS treatment is primarily directed to restore blood volume and correct
hyperosmolality. This may require the supply of intravenous fluid in the total
amount up to 8–10 liters. Therapy may be started by intravenous infusion of
saline at the rate of 1.5 liters/h for the first 2 h, followed by infusion of 0.5
liter/h of half-normal saline (0.45%) adjusted according to the clinical and
laboratory response. Insulin should also be given. This may be done according
to the small dose regimen described under DKA, although some patients may
109Clinical Emergencies in Diabetes
require larger doses. Potassium should be supplied (see under DKA) with
special attention because, in the absence of acidosis, the intracellular K
+
transfer induced by insulin administration is more pronounced. Attention
should also be paid to the possible development of infections or thrombosis
or DIC to start timely the appropriate therapy.
Suggested Reading
Foster DW, McGarry JD: The metabolic derangements and treatment of diabetic ketoacidosis. N Engl
J Med 1983;309:159–169.
Genuth SM: Diabetic ketoacidosis and hyperglycemic hyperosmolar coma. Curr Ther Endocrinol Metab

1997;6:438–447.
Gonzalez-Campoy JM, Robertson RP: Diabetic ketoacidosis and hyperosmolar nonketotic state: Gaining
control over extreme hyperglycemic complications. Postgrad Med 1996;99:143–152.
Silink M: Practical management of diabetic ketoacidosis in childhood and adolescence. Acta Paediatr
1998;425(suppl):63–66.
Siperstein MD: Diabetic ketoacidosis and hyperosmolar coma. Endocrinol Metab Clin North Am 1992;
21:415–432.
Umpierrez GE, Khajavi M, Kitabchi AE: Review: Diabetic ketoacidosis and hyperglycemic hyperosmolar
nonketotic syndrome. Am J Med Sci 1996;311:225–233.
Whiteman VE, Homko CJ, Reece EA: Management of hypoglycemia and diabetic ketoacidosis in preg-
nancy. Obstet Gynecol Clin North Am 1996;23:87–107.
F. Belfiore, Institute of Internal Medicine, University of Catania, Ospedale Garibaldi,
I–95123 Catania (Italy)
Tel. +39 095 330981, Fax +39 095 310899, E-Mail francesco.belfi
110Belfiore/Iannello
Chapter VIII
Belfiore F, Mogensen CE (eds): New Concepts in Diabetes and Its Treatment.
Basel, Karger, 2000, pp 111–124

Clinical Emergencies in Diabetes.
2: Hypoglycemia
F. Belfiore, S. Iannello
Institute of Internal Medicine, University of Catania, Ospedale Garibaldi,
Catania, Italy
Definition
The term hypoglycemia refers to a biochemical conditionresulting from an
abnormally low plasma glucose level, less than the lower value of the normal
range(50–45 mg% or 2.8–2.5 mmol/l). Thus, the term hypoglycemiaseemsinap-
propriate to define a variety of clinical manifestations associated with abnor-
mally low blood glucose and consisting of signs and symptoms of adrenergic

activation and neuroglycopenia, responsive to glucose administration.
In infants, during the first 48 h of life, hypoglycemia may occur, with
glycemic values =30 mg% or 1.7 mmol/l, with a frequency of about 10% of
live births. A brief hypoglycemic episode can cause moderate alterations of
the brain whereas prolonged hypoglycemia can cause profound dysfunctions,
tissue damage and also death of the brain. This depends on the fact that the
deposit of glycogen in brain is negligible (the reserve of energy lasts 2–3 min)
and that glucose is not synthesized by the central nervous system (CNS). Thus,
glucose (together with oxygen) is an obligate primary energy substrate for the
brain tissue and is entirely derived from the circulation. The brain tissue
utilizes 120 g/day of glucose and about 90% of total energy needed for cerebral
functions derives from glucose oxidation. The brain cannot utilize alternative
substrates (as circulating FFA) as energy fuel thus being very sensitive to
hypoglycemia. In some particular situations, at least some parts of the brain
might utilize ketoacids.
Hypoglycemia is avery uncommon event, apart from persons withdiabetes
treated with insulin or hypoglycemic drugs. The diagnosis of hypoglycemia is
based upon Whipple’s triad,i.e. hypoglycemia, symptomsof hypoglycemia, and
correction of the symptoms with the normalization of blood glucose.
111
Glucose Counterregulation
Insulin regulates glycemia through modulation of hepatic glucose produc-
tion in the postabsorptive state and glucose utilization in the postprandial
state, and it is the only hormone able to physiologically reduce glycemic
level. In catabolic states (fasting), insulin concentration falls and the levels of
counterregulatory hormones rise; in fact, hypoglycemia is capable of inducing
the release of counterregulatory hormones, including glucagon, catechola-
mines (epinephrine and norepinephrine – released both from adrenal medulla
and the sympathetic neurons), cortisol and GH. The glucagon secretory re-
sponse to hypoglycemia is largely CNS-independent whereas catecholamine,

cortisol and GH responses are prevailingly CNS-dependent. Glucagon acts
within minutes and is the primary hormone of glucose maintenance (by stimu-
lating hepatic glucose production through increase in glycogenolysis and glu-
coneogenesis). Catecholamines also act swiftly, stimulating glucose production
and limiting glucose utilization in humans through both 
2
- and 
2
-adrenergic
mechanisms. Cortisol and GH, on the contrary, act within several hours
with a delayed glucoregulatory action (antagonizing insulin action, mobilizing
substrate and activating hepatic gluconeogenesis through the induction of the
relative gluconeogenic enzymes). All these hormones have a synergic action
on the induction of hyperglycemia and on the prevention and correction of
hypoglycemia. Glucagon plays the most important counterregulatory action
whereas catecholamines play a minor role, that becomes important when there
is glucagon deficiency, as it often happens early during the course of diabetes
mellitus. Catecholamines are the warning system in hypoglycemia through the
symptoms and signs of adrenergic overactivity. Cortisol and GH play no role
in short-term hypoglycemia but have a substantial role in the recovery from
long-term hypoglycemia. The relevance of other hormones or neurotransmit-
ters in preventing and correcting hypoglycemia has been debated but it is not
definitely established. In type 1 diabetic patients, counterregulation is often
altered and, in some patients it may be very deficient. It has been reported
that almost all diabetic patients show a deficient glucagon secretory response
to hypoglycemia, perhaps as a result of the long-term hyperglycemia (glucose
toxicity) or the loss of the regulating effect of insulin on glucagon secretion.
In the presence of adefective glucagon secretion, type1 diabetic patients during
hypoglycemic episodes became dependent upon catecholamines to correct low
glycemic level, i.e. epinephrine response compensates for deficient glucagon

response. Some diabetic patients with long-standing disease have also a defi-
cient catecholamine response to hypoglycemia and this combined disorder
impairs glucose counterregulation and represents a high risk of iatrogenic
hypoglycemia in these subjects. GH and cortisol responses to hypoglycemia
112Belfiore/Iannello
Table 1. Causes of hypoglycemia
A. Fasting hypoglycemia B. Postprandial or reactive hypoglycemia
1. Reduced glucose production Alimentary hypoglycemia (gastrectomy,
Liver or renal insufficiency gastrojejunostomy, pyloroplasty
Deficiencyofcounterregulatoryhormones or vagotomy)
Childhood ketotic hypoglycemia Hyperthyroidism
(substrate or enzyme deficiency) Obesity with hyperinsulinism
Drugs (alcohol, salicylates, -blockers) Early stage of type 2 diabetes, prediabetes
or IGT
2. Increased glucose utilization
Idiopathic reactive hypoglycemia
-Cell tumor or insulinoma
Idiopathic postprandial syndrome or
Functional hypersecretion of -cells
pseudohypoglycemia
Autoantibodies to insulin
Inherited disorders of carbohydrate
Autoantibodies to insulin receptors
metabolism in children
Sepsis
Intake of leucine in leucine-sensitive
Insulin or insulin-releasing drugs
children
(sulfonylureas, pentamidine, quinine)
Newborn hypoglycemia (first hours of life,

if mother is diabetic)
Extrapancreatic non--cell tumors
Childhood nonketotic hypoglycemia
(deficit of carnitine or of enzymes of
FFA utilization
Exhaustive exercise
3. Factitious or artifactual
Factitious hypoglycemia (surreptitious
insulin or sulfonylurea administration)
Artifactual hypoglycemia (in hemolytic
anemia or in leukemia or in hyperlipemia)
in type 1 diabetes are usually not reduced, but deficiency of their secretion
may occur.
Classification of Hypoglycemia (see table 1)
Postabsorptive or Fasting Hypoglycemia
Fasting hypoglycemia may result from impaired hepatic glucose produc-
tion (involving glycogenolysis or gluconeogenesis) or enhanced peripheral
glucose utilization. It can be induced by several causes, listed below.
Reduced Glucose Production. This occurs in the following instances:
(1) Chronic failure of critical organs such as liver diseases (hepatitis,
cirrhosis or hepatoma, severe heart failure with hepatic congestion) which
113Clinical Emergencies in Diabetes
impair hepatic glucose production, or conditions of inadequate substrate store
and supply (chronic renal failure, malnutrition, starvation or cachexia, ano-
rexia nervosa, late pregnancy).
(2) Deficiency ofcounterregulatory hormones (glucagon andepinephrine,
cortisol and GH) that impairs gluconeogenesis, as occurs in hypopituitarism,
in adrenal insufficiency and rarely in glucagon deficiency.
(3) Ketotic hypoglycemia of infancy and childhood, linked to substrate
deficiency or due to defects in one or more of the gluconeogenic or glyco-

genolytic enzymes, sometimes associated to lactic acidosis.
(4) Drugs such as alcohol (which inhibits hepatic gluconeogenesis) espe-
cially when associated to fasting, salicylates (a common cause of hypoglycemia
in infants) which would increase peripheral glucose utilization and reduce
hepatic gluconeogenesis, -blockers (which reduce the glycogenolytic response
to epinephrine).
Increased Glucose Utilization. Several causes may lead to increased glucose
utilization:
(1) Endogenous hyperinsulinism (that causes glucose overutilization)
produced by: (a) -cell tumor or insulinoma (a rare, most often small and
single, benign tumor occurring in 1/250,000 adult individuals) or islet cell
hyperplasia (nesidioblastosis), a rare syndrome in adult subjects; (b) func-
tional hypersecretion of -cells; (c) autoimmune hypoglycemia (autoantibod-
ies against insulin, with inappropriate release of antibody-bound insulin in
the circulation), common in Japan; (d) rare instances of acanthosis nigricans
(insulin receptor autoantibodies, which most often cause insulin resistant
diabetes, can in some patients act as insulin-like factors); (e) ectopic insulin
secretion.
(2) Sepsis (cytokines associated to endotoxinemia increase insulin re-
lease).
(3) Insulin or drugs that stimulate insulin release, such as sulfonylurea
compounds in diabetic patients, pentamidine (which exerts a toxic effect with
-cell cytolysis), and quinine (which induces massive insulin release, although
this effect is not well demonstrated).
(4) Hypoglycemia of infants born from diabetic mothers, occurring during
the first hours of life (provoked by fetal hyperinsulinemia linked to hyperplasia
of -cells induced by maternal hyperglycemia and hyperglucagonemia).
(5) Non--cell or extrapancreatic large tumors of mesenchymal (50%) or
epithelial origin (5–10%) or hepatomas (25%) or other carcinomas (5–10%)
or some malignant hematologic diseases (5–10%), in which hypoglycemia is

induced by production of insulin-like growth factors such as IGF-2, that
interacts with insulin receptors (and may suppress endogenous insulin secre-
tion), or by overutilization of glucose (by the tumoral tissue).
114Belfiore/Iannello
(6) Nonketotic hypoglycemia due to systemic carnitine deficiency or en-
zymatic defects which limit the utilization of FFA or ketones (which entails
enhanced glucose oxidation for energetic purposes).
(7) Prolonged and exhaustive exercise, especially in untrained persons
(increased glucose utilization).
Factitious or Artifactual Hypoglycemia. Two conditions should be dis-
tinguished:
(1) Factitious hypoglycemia from deliberate and surreptitious insulin or
sulfonylurea assumption (especially in medical people or family members of
diabetic patients with psychiatric disturbances).
(2) Artifactual hypoglycemia as it may occur in hemolytic anemia or in
leukemia and leukemic reactions (due to overutilization of glucose in the test
tube by young erythrocytes or leukemic leukocytes) or in the presence of
marked hyperlipemia (which may cause a 15% – or more – underestimation
of glucose concentration).
Postprandial or Reactive Hypoglycemia
This form of hypoglycemia occurs within 6 h after a meal, and includes
several forms, listed below:
(1) Alimentary hypoglycemia (or alimentary hyperinsulinism) caused by
gastrectomy, gastrojejunostomy, pyloroplasty or vagotomy, involving about
5–10% of operated patients and developing 30–120 min after ingestion of
carbohydrate-containing meals (due to rapid gastric emptying and glucose
absorption which stimulate excessive insulin release, and perhaps also to hyper-
secretion of enterohormones such as enteroglucagon, secretin, GIP, etc.); it
may perhaps also occur in patients with hyperthyroidism, or in obesity with
hyperinsulinism.

(2) Early stage of type 2 diabetes or prediabetes or IGT (deficient early-
phase insulinrelease leadsto higherglucose elevation with subsequent excessive
stimulation of insulin secretion). However, it should be mentioned that the
relationship between the early stage of type 2 diabetes or prediabetes or IGT
and postprandial hypoglycemia is not well established.
(3) Idiopathic reactive hypoglycemia or true hypoglycemia (with lowered
glucose levels), a rare syndrome characterized by adrenergic symptoms without
symptoms of severe neuroglycopenia, probably linked to an increased insulin
response or a higher affinity of insulin receptors or to a subtle dysfunction of
gastrointestinal tract.
(4) Idiopathic postprandial syndrome or pseudohypoglycemia (with a
near-normal glycemic value), characterized by adrenergic symptoms and
light symptoms of neuroglycopenia, which develop regularly and repetitively
during the patient’s life (causes are unknown and might include enhanced
115Clinical Emergencies in Diabetes
Table 2. Clinical signs and symptoms of hypoglycemia
Sympathetic/parasympathetic activation Neuroglycopenia
A. Clinical signs and symptoms of adrenergic Clinical signs and symptoms of
activation neuroglycopenia
Pallor, tremor, palpitations and anxiety Headache, dizziness, fatigue, irritability or
Acute sensation of hunger apathy and lethargy
Occasionally hypothermia, vomiting, Frequent yawning and perioral numbness
fever, moderate tachycardia, crises of Disturbed vision and diplopia
systolic hypertension Paresthesias and motor dysfunction
B. Clinical signs and symptoms of
Cognitive impairment, mental confusion
and inebriation
parasympathetic activation
Personality changes, psychotic behavior
Nausea and eructation

Occasionally transient hemiparesis or
Cold sweating
focal neurologic deficits
Mitigation of expected tachycardia or true
Convulsions (in children simulating true
bradycardia
crises of epilepsy)
Mild hypotension
Semi-coma, coma and even death
epinephrine release in some subjects, with stress or anxiety contributing in
many subjects).
(5) Inherited disorders ofcarbohydrate metabolism in children (hereditary
fructose intolerance from deficiency of fructose-1-P aldolase or galactosemia
from deficiency of galactose-1-P uridyltransferase).
(6) Intake of leucine in leucine-sensitive children (due to increased insulin
secretion).
Clinical Signs and Symptoms of Hypoglycemia (see table 2)
The clinical manifestations of hypoglycemia are generally nonspecific and
varying,notonlyfrompatienttopatientbutalsointhesamesubjectfromepisode
to episode. Their development can depend not only on the glycemic value but
also on the rate of the fall in blood glucose. Manifestations can be distinguished
into adrenergic (due to sympathetic activation) and neuroglycopenic (due to
neuronal alterations secondary to glucose deprivation). When glucose drops
rapidly, adrenergicsymptoms are most evident whilewhenglucose drops gradu-
ally neuroglycopenic symptoms may dominate the clinical picture. During a hy-
poglycemic episode, the response of counterregulatory hormones begins before
the symptomatic glucose threshold is reached.
116Belfiore/Iannello
Neuropenic symptoms may not occur even in the presence of glucose
level as low as 25–30 mg/dl (1.4–1.7 mmol/l) due to the ability of normal

persons to increase brain blood flow and therefore glucose delivery. This
adaptation may be prevented in patients with cerebral atherosclerosis and
inelastic vessels, in whom neuropenic symptoms may appear at relatively high
glucose levels.
It should be pointed out that severe hypoglycemic reactions may occur
even in the presence of near-normal or even high glycemic values (pseudohypo-
glycemia), especially in diabetic patients; on the other hand, there may be no
clinical hypoglycemic reactions with very low concentrations of plasma glucose
(25–30 mg% or 1.4–1.7 mmol/l). The most important factors probably are the
rate of fall in glycemia and the fact that the glucose plasma level may not
strictly reflect the glucose concentration in brain tissue. A glycemic range
(55–70 mg% or 3.00–3.88 mmol/l) seems to exist in which dysfunction from
neuroglycopenia and activation of counterregulatory hormones occur but
symptoms are not yet manifest; therefore, the value of 3.88 mmol/l may be a
cut-off value of hypoglycemia, useful and safe to consider in the treatment of
diabetes mellitus.
Adrenergic Symptoms and Signs
These are due to catecholamine hypersecretion that develops in response
to a blood glucose level =53 mg% or 2.95 mmol/l, and include pallor, anxiety,
tremor, palpitations, tachycardia (occasionally with crises of systolic hyperten-
sion) and acute sensation of hunger. It is noteworthy that symptoms and signs
induced by parasympathetic response can also occur during hypoglycemia,
producing nausea, eructation, cold sweating, mitigation of expected tachy-
cardia or true bradycardia, and mild hypotension.
Neuroglycopenic Symptoms and Signs
These are due to dysfunction of CNS that develops in response to hypogly-
cemia =45 mg% or 2.50 mmol/l, and include headache, dizziness, fatigue,
irritability or apathy, lethargy, frequent yawning, cognitive impairment, mental
confusion, inebriation, personality changes and psychotic behavior, disturbed
vision and diplopia, perioral numbness, paresthesias, motor dysfunction, con-

vulsions, occasionally transient hemiparesis or focal neurologic deficits (espe-
cially in elderly diabetic patients), semi-coma, complete loss of consciousness
until hypoglycemic coma and even death. The different neurologic manifesta-
tions have been correlated with specific sites of the brain involved in different
degrees of hypoglycemia. Clinical hypoglycemic symptoms and signs some-
times suggest true mental disorders, accounting for the frequent reported
mistake or delay in diagnosis.
117Clinical Emergencies in Diabetes
In children, adrenergic manifestations are near-absent and neurogly-
copenic symptoms can predominate with seizures simulating true crises of
epilepsy. A failure to develop several adrenergic symptoms before the develop-
ment of neuroglycopenic symptoms (hypoglycemia unawareness) is observed
in 50% of patients with long-standing diabetes (due to the reduced response of
sympathetic system to hypoglycemia, secondary to the autonomicneuropathy).
However, glucose threshold may also be lowered by hypoglycemia itself which
may cause subsequent hypoglycemia unawareness, as it may be observed in
patients with insulinoma.
Hypoglycemia Induced by Insulin or Sulfonylurea Treatment
The commonest form of hypoglycemia is that induced by insulin or sul-
fonylureas as well as by ethanol. It accounts for about 60% of patients hospital-
ized for hypoglycemia, while renal disease accounts for 15%, liver diseases for
15% and malnutrition for 10%. In diabetic patients, hypoglycemic episodes
can be isolated or recurrent, and are due to a mismatch of insulin or sulfonylu-
rea therapy to meal pattern or physical activity. In long-standing diabetes, a
defective counterregulation with deficiency of glucagon and epinephrine may
contribute; these subjects are at 25-fold increased risk for severe iatrogenic
hypoglycemic crises during intensive insulin treatment.
In type 1 diabetics, mild to moderate symptomatic hypoglycemic crises
occur in about 90% of patients (frequently during the night). In insulin-treated
diabetics, severe hypoglycemia or hypoglycemic coma was reported in 9–10%

of cases in 1 year during conventional insulin therapy, and higher figures most
probably apply for patients treated with intensive insulin regimen.
The most serious hypoglycemic episodes in diabetic patients can happen
with the sulfonylurea compounds. They may occur at any time after ingestion
of the drug (from 30–60 min to many hours later) and are characterized by
diminished or absent autonomic signs, prolonged or relapsing hypoglycemia
and by a response to glucose which is not as prompt as in insulin-induced
hypoglycemic episodes.
Predisposing factors may hasten the onset or increase the intensity of the
hypoglycemic effects of insulin or sulfonylureas (table 3). It is important to
avoid hypoglycemia in a diabetic mother in the early period of gestation
because maternal hypoglycemia can cause malformations, for a detrimental
effect on growth and differentiation of the fetus.
Hypoglycemia canhave unfavorable long-or short-term effects on vascular
complications of diabetes. In fact, it increases systolic and diastolic pressures,
glomerular filtration rate, viscosity and platelet aggregation. Repeated episodes
118Belfiore/Iannello
Table 3. Predisposing factors in drug-induced diabetic hypoglycemia
Undernutrition or omission of food or Administration of a -blocker
starvation Defective counterregulation, hypoglycemia
Unexpected exercise unawareness
Renal or hepatic dysfunction Lowered glycemic threshold for
Abuse of alcohol hypoglycemia and counterregulation
Acute sickness during intensive insulin treatment
Erroneous high insulin or sulfonylurea doses (with compromised recognition of
Increased absorption of insulin from the site developing hypoglycemia)
of injection
of hypoglycemia can result in neuropsychologic deficits, especially in younger
patients (EEG changes and cognitive impairment) and, if severe, hypoglycemia
can be fatal. Related to hypoglycemia is the Somogyi phenomenon, which is

a posthypoglycemic hyperglycemia that most often follows a nocturnal fall in
blood glucose level (O50 mg/dl) and is due to the response of counterregu-
latory hormones and the subsequent increase in glycemia. It can be contrasted
by reducing the evening doses of drugs. The Somogyi phenomenon should be
distinguished from the morning hyperglycemia which may be seen in insulin-
treated patients, named ‘dawn phenomenon’, linked to the increase in GH
secretion normally associated with sleep.
Diagnosis
Hypoglycemia in a diabetic patient taking insulin or sulfonylurea drugs
is not a diagnostic problem, especially considering that clinical symptoms are
most characteristic. Instant glycemic determination (which can be obtained
also with a self-blood glucose monitoring device) confirms or excludes the
diagnosis of hypoglycemia. In hypoglycemic children, seizures are common
and may simulate epilepsy (on the other hand, hypoglycemia may favor or
trigger an epileptic focus). An EEG performed successively may help in the
diagnosis.
Diagnostic problems exist in nondiabetic subjects with symptoms of hy-
poglycemia because of the several possible causes (factitious or reactive hy-
poglycemia, insulinoma, extrapancreatic tumors, etc.). In the presence of a
patient with suspicion of hypoglycemia, itis crucialto demonstrate the hypogly-
cemia with a specific determination of blood glucose in specimens obtained
during the hypoglycemic event. Very useful in the diagnosis of hypoglycemia
is also the assessment of levels of plasma insulin, C-peptide, counterregulatory
119Clinical Emergencies in Diabetes
Table 4. Diagnostic tests for hypoglycemia
Simultaneous determination of glycemia Tolbutamide test
and insulinemia during hypoglycemic Glucagon test
episodes Vigorous exercise during fasting
Supervised fasting (the gold standard test) Proinsulin determination
C-peptide suppression test Leucine test

Sulfonylurea assay in plasma or urine Search for antibodies to insulin
5h-OGTT Search for autoantibodies against insulin or
Insulin tolerance test insulin receptors
hormones, drugs or alcohol. It is important to have an accurate history of
the patient to distinguish between fasting or postprandial hypoglycemia and to
relate hypoglycemic episodes and symptoms and signs. The clinical evaluation
should refer to weight loss (extrapancreatic tumor or endocrine deficits) or
weight gain (insulinoma or reactive hypoglycemia), presence of autoantibodies
against insulin receptors (acanthosis nigricans), hepatomegaly (galactosemia
or glycogenosis), and presence of tumoral abdominal masses (of mesenchymal
or epithelial origin) localized by CTor abdominal sonography. Ifan insulinoma
is suspected, the tumor should be localized before surgery with ultra-
sonography (also used intraoperatively), celiac angiography or CT evaluation.
Several diagnostic tests can be very helpful (table 4), and are outlined
below:
(1) Simultaneous determination of glycemia and insulinemia during the
hypoglycemic episode and at fasting on 3 consecutive days.
(2) Supervised fast, which consists of simultaneous determinations every
4–6 h of glycemia, insulinemia and C-peptide during fasting periods of 24,
48 and 72 h. In insulinoma, glycemia falls while C-peptide and insulinemia
remain near-unmodified. Quantitation of plasma cortisol, FFA, glucagon and
total ketones can sometimes be useful. In normal male individuals, mean
glucose at 72 h is 3.4–3.9 mmol/l (or 62–71 mg%) and in normal female in-
dividuals is 2.7–2.9 mmol/l (or 48–52 mg%) while mean insulin is 6 and
4 U/mL, respectively. A diagnosis of hypoglycemia is probable with values
of glycemia =2.5 mmol/l (or 45 mg%) and presence of symptoms which are
rapidly relieved by administration of glucose. In the presence of hyperinsuline-
mia and increase of C-peptide an insulinoma should be suspected, while in
the presence of hyperinsulinemia with a low level of C-peptide the possibility
of factitious hypoglycemia induced by exogenous insulin (with suppression of

C-peptide secretion) should be considered. On the other hand, in factitious
hypoglycemia induced by sulfonylureas, both plasma insulin and C-peptide
levels are increased.
120Belfiore/Iannello
(3) C-peptide suppression test, based on the fact that suppression of
the release of endogenous insulin and C-peptide during insulin infusion
(0.125 U/kg over 1 h or over 3 h) is impaired in about 90% of patients with
insulinoma.
(4) Assay of sulfonylurea compounds in plasma or urine to diagnose
factitious hypoglycemia induced by these drugs.
(5) Five-hour oral glucose tolerance test (5h-OGTT), useful for the diag-
nosis of reactive hypoglycemia in about 50% of cases (it is not a specific test,
as it may be positive both in normal persons and in subjects with pseudohypo-
glycemia).
(6) Insulin tolerance test (0.1 U/kg intravenous insulin and determina-
tions of ACTH, cortisol, epinephrine, norepinephrine, GH and glucagon),
useful in cases of counterregulatory hormone deficiencies.
(7) Intravenous tolbutamide infusion (1 g over 3 min), positive in about
80% of the cases with insulin-secretory tumors (induces a severe hypoglycemia,
with average value of glycemic levels at 120, 150 and 180 min O55 mg/dl in
lean and O62 mg/dl in obese subjects).
(8) Glucagon test (1 mg intravenous glucagon should produce an insulin
peak P130 U/ml), useful in glycogenosis (in this disease, however, enzyme
determinations in liver and muscle biopsy are the best diagnostic test).
(9) Vigorous exercise in fasted state will provoke hypoglycemia in insu-
linoma patients (this is not a well-standardized test).
(10) Proinsulin determination (?25% of total insulinemia in about 85%
of patients with insulinoma, compared to 10–15% of the normal subjects; this
proinsulin excess is lacking in factitious hypoglycemia).
(11) Leucine test (intravenous L-leucine infusion, 200 mg in 30 min, or

an oralload of0.15 g/kg, with serial samples obtained for 1 or 2 h, respectively),
that evokes an excessive insulin response in children susceptible to leucine and
in about 70% of patients with insulinoma.
(12) Search for antibodies to insulin (if present in persons not receiving
insulin therapy can indicate a surreptitious insulin use) or for autoantibodies
against insulin or insulin receptors (useful to diagnose acanthosis nigricans
and early stage of insulin-resistant diabetes).
Treatment
Hypoglycemia in Diabetic Patients
The treatment of hypoglycemia depends upon the cause and the severity
of the hypoglycemic episode. Diagnosis should be made promptly to select
promptly the appropriate treatment. Commonly, episodes of hypoglycemia
121Clinical Emergencies in Diabetes
happen in diabetic patients and are treated providing exogenous glucose or
stimulating endogenous glucose production with glucagon. When possible,
glycemic concentration should be measured before glucose treatment, but this
should not be the cause of delay of treatment. Mild to moderate hypoglycemia
in conscious and alert patients can be treated orally with foods containing
20–30 g of carbohydrates, eventually followed, after 20 min, by a second
smaller assumption of 10–20 g carbohydrates. Suitable foods include honey,
sweetened drink, cola beverages, syrups, fruit juices, candies, etc. In uncon-
scious patients with serious hypoglycemia producing confusion, semi-coma or
coma (who is not cooperative and cannot take oral feedings), the obligate
treatment is intravenous glucose administration as a bolus of about 25 or 50 g
(as 33–50% hypertonic glucose solution) followed by a 5–10% dextrose infusion
in order to prevent relapse of hypoglycemia, while monitoring frequently the
glycemic values during the acute hypoglycemic phase. An effective therapy is
also the prompt intravenous glucagon injection (1–2 mg, repeated in 5 min if
necessary; in children 0.5–1 mg), even if the time of recovery is longer
(6–20 min) than following intravenous glucose (5–10 min). A subcutaneous

or intramuscular injection of glucagon is recommended when intravenous
administration is not possible or in outpatient treatment, even if the obtained
glycemic response is transient and moderate. When a normal consciousness
is recovered, oral glucose or carbohydrates can be provided. Glucagon is the
ideal agent (safe and efficacious) to keep in medical bags or for use by the
family members of the diabetic patient. Glucagon (that mobilizes glucose from
liver by stimulating glycogenolysis) has a poor effect or is ineffective in starved
or undernourished or uremic or hepatologic or inebriated patients (due to
deficiency of hepatic glycogen stores) and in chlorpropamide-induced hypogly-
cemia (the drug would inhibit glycogenolysis). Adrenaline is rarely used
(0.5–1 ml, subcutaneously, repeated eventually after 15 min if no response is
observed). Some hypoglycemic patients may require large amounts of glucose
to maintain normoglycemia (20–30% dextrose solutions). Sulfonylurea-
induced hypoglycemia may last for a prolonged time, until 72 h (probably
hepatic orrenal diseasesand interactions withother hypoglycemia-potentiating
drugs may play a role in this prolonged effect). In these patients, a constant
infusion of hypertonic glucose should not be discontinued until the recovery
from hypoglycemia is definitely complete, and clinical surveillance should be
continued to notice possible relapse. In patients with prolonged hypoglycemia
or retarded recovery from hypoglycemia (particularly if hyperazotemic),
100 mg of hydrocortisone in intravenous bolus may be provided (repeated
eventually after 4 h), to facilitate glucose entry into neural cells and to induce
gluconeogenesis (hydrocortisone, however, should never be used as the sole
treatment in a severe hypoglycemic event or in coma).
122Belfiore/Iannello
Precautionary Measures
In patients at risk for hypoglycemia (diabetics treated with insulin or
hypoglycemic drugs, alcoholists, etc.) several precautionary measures must be
considered to prevent and minimize hypoglycemic episodes such as adjusted
therapeutic regimens, relatively high glycemic goals, etc. In this regard, The

Guidelines for Diabetes Care of the European Diabetes Policy Group of IDF
(1998) suggest the following principles. Recurrent hypoglycemia at a particular
time or times of day implies a mismatch of insulin therapy to meal pattern
and/or physical activity. Therefore, it should be reviewed whether a repeated
change in meal or activity behavior has occurred, and bear in mind the possibil-
ity of changes in underlying insulin sensitivity (age, renal, endocrine). On this
basis, the opportunity of specific insulin dose adjustment should be taken
into account. In cases of erratic hypoglycemia, the possible causes should be
assessed, such as missed or varied meals or snacks, wrong physical activity,
abuse of alcohol, injection site abnormalities, errors in insulin doses, gastropa-
resis, etc. The possibility should be considered of undetected nighttime or
other hypoglycemia (especially if HbA
1c
is lower than average) and the insulin
doses or food intake should be modified accordingly (avoiding any glucose
excursion to =70 mg/dl or 4 mmol/l). It is especially useful to provide educa-
tion and training in recognizing early cognitive dysfunction for patients and
to advise caution over driving. In nocturnal hypoglycemia, it should be recom-
mended to take the evening NPH insulin and a slowly absorbed carbohydrate
snack as late as possible, and to use a rapid-acting insulin analogue before
the main evening meal.
Hypoglycemia in Insulinoma and Extrapancreatic Tumors
In insulinoma patients, surgery is the elective treatment, while medical
treatment is indicated only in the presurgical phase (diazoxide in doses of
300–1,200 mg/day, per os, and octreotide in doses of 100–600 g/day, sub-
cutaneously). Treatment of nonpancreatic insulin-producing tumors is very
difficult and unsatisfactory (drugs suggested: streptozotocin plus doxorubicin
or fluorouracil, or chlorozotocin).
Other Forms of Hypoglycemia
In hypoglycemia from intolerance to fructose or galactose, it is mandatory

to prescribe a diet low in these sugars. In the intolerance to leucine, a diet
deprived of milk (which is rich in leucine) is useful and should be started as
soon as possible to prevent the precocious brain damage.
Therapy of other forms of hypoglycemia includes hormone replacement
in patients with deficiency of counterregulatory hormones and dietary adjust-
ments in reactive hypoglycemia and pseudohypoglycemia (such as avoidance
123Clinical Emergencies in Diabetes
of prolonged fasting or simple sugars assumption, restriction of caloric intake,
reduction of meal size, consumption of high-protein low-carbohydrate meals
and snacks, diets high in fibers, use of -adrenergic blockers, supply of vitamins
and minerals, etc).
Conclusion
In conclusion, hypoglycemia is a serious problem in diabetic patients
(especially type 1 diabetics) that requires a careful monitoring because it causes
morbidity and, directly or indirectly (falls, driving accidents, drowning, status
epilecticus, etc.), an increased mortality. It is preventable with an adequate
education of both the diabetologist and the patient. The best treatment of
hypoglycemia is prevention and, to this end, it is necessary to develop methods
to deliver insulin in a physiological manner and to bear in mind that strict
euglycemia is not an appropriate objective in diabetic patients with defective
counterregulation (to avoid hypoglycemia). Patient education, medical sup-
port, self-glucose monitoring and appropriate insulin regimens are helpful
to maintain a good glycemic control while minimizing severe hypoglycemic
episodes in diabetic patients.
Suggested Reading
American Diabetes Association: Clinical practice recommendations 1997. Diabetes Care 1997;20(suppl 1):
1–70.
Cryer PE, Fisher JN, Shamoon H: Hypoglycemia. Diabetes Care 1994;17:734–755.
Cryer PE, Gerich JE: Hypoglycemia in insulin dependent diabetes mellitus: Insulin excess and defective
glucose counterregulation; in Rifkin H, Porte D (eds): Diabetes mellitus. Theory and Practice, ed 4.

New York, Elsevier, 1990, pp 526–546.
Frier BM, Fisher BM, Gray CE, Beastall GH: Counterregulatory hormonal responses to hypoglycemia
in type 1 (insulin-dependent) diabetes: Evidence for diminished hypoglycemic-pituitary hormonal
secretion. Diabetologia 1988;31:421–429.
Gerich JE, Langlois M, Noacco C: Lack of glucagon response to hypoglycemia in diabetes: Evidence for
an intrinsic pancreatic -cell defect. Science 1973;182:171–173.
Grunberger G, Weiner GL, Silverman R, Taylor S, Gorden P: Factitious hypoglycemia due to surreptitious
administration of insulin: Diagnosis, treatment, and long-term follow-up. Ann Intern Med 1988;
108:252–257.
Service FJ: Hypoglycemic disorders. N Engl J Med 1995;332:1144–1152.
F. Belfiore, Institute of Internal Medicine, University of Catania, Ospedale Garibaldi,
I–95123 Catania (Italy)
Tel. +39 095 330981, Fax +39 095 310899, E-Mail francesco.belfi
124Belfiore/Iannello
Chapter IX
Belfiore F, Mogensen CE (eds): New Concepts in Diabetes and Its Treatment.
Basel, Karger, 2000, pp 125–134

Mechanisms of Diabetic Complications
(Nephropathy) as Related to
Perspectives of Treatment
Mark E. Cooper
Department of Medicine, University of Melbourne, Austin & Repatriation Medical
Centre (Repatriation Campus), West Heidelberg, Vic., Australia
Introduction
Diabetic nephropathy (DN) is characterized by a number of functional
and structural abnormalitites. Functional changes include initial renal hyper-
filtration/hyperperfusion with subsequent development of microalbuminuria
which is a modest increase in the urinary excretion of albumin and is not
detected by conventional dipstick methods. At this stage, ultrastructural

changes including glomerular basement membrane thickening, glomerular
hypertrophy and mesangial expansion are present. This is followed by the
subsequent development of glomerulosclerosis and tubulointerstitial fibrosis.
Overt proteinuria supervenes followed by the development of renal impairment
and ultimately renal failure. Although the renal complications of diabetes had
already been described in the 18th century, it is only over the last 20 years
that the mechanisms linking chronic hyperglycemia to the development of DN
have begun to be unravelled (fig. 1). It is likely that DN occurs at least partly
as a result of a chronic glucose-dependent process.
Glycation
In diabetes, a state of chronic hyperglycemia, there is an acceleration of
the Maillard or browning reaction. This is a spontaneous reaction between
glucose and proteins, lipids or nucleic acids, particularly on long-lived proteins
such as the collagens. There is a sequence of biochemical reactions, many of
125
Fig. 1. Schema depicting potential interactions between metabolic and hemodynamic
pathways in the pathogenesis of DN. The crosses represent potential targets for intervention.
1>Improved glycemic control, e.g. intensified insulin therapy; 2>inhibitors of advanced
glycation formation, e.g. aminoguanidine; 3>cross-link breakers, e.g. PTB, ALT-711; 4>
inhibitors of polyol formation, e.g. aldose reductase inhibitors; 5>inhibitors of PKC, e.g.
LY-333531; 6>inhibitors of vasoactive hormone formation/action, e.g. ACE inhibitors, AII
and ET receptor antagonists; 7>inhibitors of cytokine formation/action. See text for abbrevi-
ations.
which are still poorly defined, leading to the formation of a range of advanced
glycation end-products (AGEs). These modified long-lived tissue proteins are
formed as a result not only of glycation but also oxidative processes and many
of theseAGEs are now considered glycoxidation products. Over the last decade,
an increasing number of AGEs have been identified. However, the identity of
the AGEs linked to diabetic complications and in particular to renal disease
has not been clearly determined. Various antibodies to AGEs have now been

developed and using a variety of immunohistochemical techniques, increased
AGE levels have been reported in both human and experimental diabetes. Our
own group using a radioimmunoassay has detected increased AGE levels in
the diabetic kidney and using immunohistochemistry we have localized this
increase in AGE levels to both the glomerulus and tubulointerstitium.
Over the last few years, a number of AGE-binding sites have been identi-
fied. The first binding site to be cloned has been termed RAGE, this protein
having been detected by immunohistochemistry by our group in the kidney,
primarily in distal tubules and to a lesser extent in glomeruli. It has been
126Cooper
suggested that RAGE has a central role in the development of vascular disease
in diabetes by influencing various pathological processes including expression
of adhesion molecules involved in mononuclear cell recruitment and hyperper-
meability. At least three other proteins which bind to AGEs have recently been
cloned. It has been postulated that these proteins may mediate a range of
functions including clearance of AGEs and activation of intracellular messen-
gers such as protein kinase C. These AGE-binding sites have been identified
in cultured mesangial cells. It is still uncertain whether these AGE-binding
proteins act primarily to clear AGEs which would be viewed as a beneficial
effect or whether they are mainly involved in activating a range of pathological
processes which lead to diabetic complications.
The importance of the glycation pathway is under further investigation
with pharmacological inhibitors of AGE-dependent pathways having now
been developed. Aminoguanidine, which prevents AGE formation, has been
shown in experimental models of diabetes to not only reduce tissue AGE
levels, but also to retard the development of nephropathy and other diabetic
microvascular complications. In the diabetic rat, aminoguanidine retarded the
development of albuminuria and mesangial expansion. These experimental
studies have confirmed that renal accumulation of AGEs in diabetes could be
prevented by aminoguanidine treatment. Preliminary results from the Action

1 study suggest that aminoguanidine may have renoprotective effects in man.
In this large study in type 1 diabetic patients with overt nephropathy, many
of whom were already receiving ACE inhibitor therapy, aminoguanidine treat-
ment was associated with less proteinuria, less decline in creatinine clearance,
lower blood pressure and an improvement in various lipid parameters. It
remains to be determined if these effects will ultimately be translated to post-
ponement or prevention of end-stage renal failure.
Since aminoguanidine also inhibits other biochemical pathways and in
particular acts as an inhibitor of inducible NO synthase, it has been difficult
to determine if organ protection conferred by this agent is primarily via its
action as an inhibitor of AGE formation. New, potent and more specific
inhibitors of advanced glycation have been developed recently. Two of these,
ALT-462 and ALT-486, are approximately 5 and 20 times, respectively, more
potent than aminoguanidine in their ability to inhibit fluorescence generated
on reaction of lysozyme with ribose and both are approximately 20 times as
potent as aminoguanidine in preventing diabetes-related decreases in rat tail
collagen solubility in vivo. Our own group has evaluated 2,3-diaminophena-
zine, another inhibitor of AGE formation, and observed that this compound
is a potent inhibitor of renal AGE accumulation.
More recently, a new class of agents known as the thiazolium compounds
such as phenacylthiazolium bromide (PTB) have been considered as agents
127Mechanisms of Diabetic Complications
which may ultimately inhibit advanced glycation-induced tissue injury. These
agents react with and cleave covalent, AGE-derived protein cross-links. Re-
cently, another cross-link breaker, ALT-711, has been reported to improve
vascular compliance in diabetic rats. If PTB or related compounds can be
shown to be effective in the kidney, this would provide a conceptual basis for
the reversal of AGE-mediated tissue damage, which till now has been regarded
as irreversible.
Polyol Pathway

Aroleforthehyperglycemia-inducedaccelerationofpolyolpathwaymetab-
olisminmediatingthedevelopmentofDN hasbeensuggestedbysomeinvestiga-
tors.In tissues where glucoseuptakeis independent ofinsulinsuchas the kidney,
hyperglycemia results in increased levels of tissue glucose. The excess glucose
is subsequently reduced to sorbitol by the NADPH-dependent enzyme aldose
reductase, the first enzyme in the polyol pathway. The increased formation and
accumulation of sorbitol in these tissues is accompanied by a depletion of free
myoinositol, loss of Na
+
,K
+
-ATPase activity, and increased consumption of
the enzyme cofactors NADPH and NAD
+
, leading to changes in cellular redox
potential. These metabolicderangements have beenpostulated to result incellu-
lar dysfunction and, ultimately, themorphological lesions that characterize dia-
betic nephropathy. There are some experimental data suggesting that increased
polyol pathway metabolism mediates the loss of renal vascular tone in animals
with chronic hyperglycemia. Several investigators have demonstrated that glo-
merular hyperfiltration in diabetic rats could be prevented by treatment with
aldose reductase inhibitors. Long-term experimental studies have been con-
flicting with respect to effects not only on albuminuria but also on glomerular
structural injury. Indeed, although inhibitors of this pathway such as aldose
reductase inhibitors have now been available for over 20 years, clinical studies
on the role of these agents in the prevention and treatment of diabetic neph-
ropathy have been rather disappointing. Hopefully, with newer agents which
havebetterpharmacokineticsandtissuepenetration,aldosereductaseinhibitors
willplayamoreimportantroleinthe preventionandtreatmentofdiabeticmicro-
vascular complications including nephropathy.

Protein Kinase C
The adverse effects of hyperglycemia have been attributed to activation
of protein kinase C (PKC), a family of serine-threonine kinases that regulates
128Cooper
diverse vascular functions, includingcontractility, blood flow, cellularprolifera-
tion and vascular permeability. PKC activity, especially the membrane-bound
form, is increased in the retina, aorta, heart and renal glomeruli of diabetic
animals, probably because of an increase in de novo synthesis of diacylglycerol,
a major endogenous activator of PKC. It has been postulated that there is
preferential activation of the 
2
isoform of PKC in diabetes. This led to the
synthesis of anorally effective PKC--selectiveinhibitor, LY-333531. LY-333531
is a competitive, reversible inhibitor of PKC-
1
and PKC-
2
, with a 50-fold
lesser effect on other PKC isoenzymes. In studies over 2–8 weeks in diabetic
rats, LY-333531 ameliorated glomerular hyperfiltration, albuminuria and renal
transforming growth factor- overexpression. Further studiesincluding clinical
trials are now in progress with this compound.
Hemodynamic Factors
Diabetes is associated with elevations of both glomerular filtration rate
(GFR) and renal plasma flow (RPF). To explore the underlying intrarenal and
in particular intraglomerular hemodynamic changes associated with diabetes,
Hostetter and Brenner used micropuncture techniques. The major findings in
these landmark studies included the observation of elevated intraglomerular
pressure, related to relative afferent versus efferent arteriolar vasodilation in
experimental diabetes.

Renin-Angiotensin System
It has been suggested that these intraglomerular hemodynamic abnormali-
ties are due to altered vasoactive hormone action. This would imply an imbal-
ance in the actions of vasoconstrictors and vasodilators in the diabetic kidney
and has resulted in a large body of research focusing on a range of vasoactive
hormones and their receptors in the genesis not only ofthe initialhemodynamic
abnormalities but also on the subsequent glomerular ultrastructural injury.
The system most extensively investigated is therenin-angiotensin system (RAS)
which involves a series of enzymatic reactions leading to the production of
the effector peptide, angiotensin II (AII). This hormone has a diverse range
of actions including vasoconstriction, stimulation of sodium reabsorption and
of particular interest trophic effects on a range of cells including mesangial
cells. These actions are considered relevant to the postulated mode of action
of agents which interrupt the RAS such as ACE inhibitors and AII receptor
antagonists. All components of the RAS are present in the kidney, consistent
129Mechanisms of Diabetic Complications
with the view that this hormone system not only acts as an endocrine system
but can also act in a paracrine/autocrine manner.
Evidence of a role for the RAS in the genesis of diabetic complications
has been provided by arange of studies using different experimental techniques.
Although it was initially considered that diabetes was associated with a sup-
pressed RAS, primarily based onstudies assessing the systemic RAS, Anderson
et al. have identified sites of local activation of the RAS within the kidney
including the glomerulus and other renal vessels. Indeed, in a series of experi-
ments using molecular biological and immunohistochemical techniques in an
animal model of renal disease, the subtotal nephrectomy model, which has
many functional and structural similarities to diabetic nephropathy, our group
has shown that with renal injury there is de novo expression of various compo-
nents of the RAS including renin and AII within the kidney. This local activa-
tion of the RAS particularly in the proximal tubule may be particularly

important as a potential mechanism for the development of tubulointerstitial
fibrosis in advanced diabetic nephropathy. Similar changes have recently been
observed by our group in an animal model of advanced DN.
To further explore the role of the RAS in the evolution of DN, diabetes
has been induced in transgenic Ren 2 rats, a rat strain generated by insertion
of the mouse renin Ren 2 gene into their genome. This hypertensive strain
has elevated prorenin levels and the induction of diabetes leads to the rapid
development of glomerulosclerosis, tubulointerstitial injury and renal impair-
ment which can be attenuated by ACE inhibition. This provides further evi-
dence for a role for the RAS, particularly at the local level, in mediating renal
injury in diabetes.
The importance of the RAS is of particular relevance to the management
of DN. Agents which interrupt the RAS such as ACE inhibitors and AII
receptor antagonists have been shown to attenuate the development of experi-
mental DN. These agents normalize intraglomerular pressure, suppress renal
cytokine production and prevent extracellular matrix accumulation. These
beneficial effects observed in rodents have also been reproduced in man. In
various phases of human DN in the presence or absence of systemic hyperten-
sion, ACE inhibitors have been shown to reduce urinary albumin excretion
and retard the decline in GFR. Whether these drugs are superior to other
classes of antihypertensive agents remains controversial. However, a number
of studies have suggested that ACE inhibitors have renoprotective effects inde-
pendent of blood pressure reduction. The status of AII antagonists as renopro-
tective agents is not as well established with several large clinical studies using
these agents now in progress.
130Cooper
Other Vasoactive Hormones
More recent studies have suggested that other vasoactive hormone systems
including the natriuretic hormones such as atrial natriuretic peptide, the kal-
likrein-kinin system and the potent vasoconstrictor, endothelin (ET), may

also have important roles in the genesis of the hemodynamic and trophic
abnormalities which are observed inorgans undergoing diabetic vascular injury
such as the kidney. ET antagonists have been reported by several groups to
be useful in experimental DN. However, the role of ET antagonists in man
in the context of diabetes has not yet been explored. Another class of new
drugs to consider are the dual metallopeptidase or vasopeptidase inhibitors.
These agents have multiple effects on vasoactive hormone formation and
degradation. In a recent study by our group using an agent which not only
inhibits ACE but also inhibits the enzyme, neutral endopeptidase (NEP), an
enzyme involved in the degradation of vasodilators such as ANP and kinins,
it could be demonstrated that this agent reduced blood pressure and was very
potent at preventing the development of albuminuria in diabetic, hypertensive
rats. The long-term functional and structural effects of these agents in experi-
mental DN and ultimately in man are awaited with interest.
Since nitric oxide (NO) is a potent renal vasodilator, it has been suggested
that this molecule may be in excess in the diabetic kidney. Indeed, metabolites
of NO, nitrate and nitrite, are excreted in excess in the urine of diabetic rodents.
Furthermore, several groups have documented that the nonselective inhibitor
of NO synthase, L-NAME, can reduce the elevated GFR and RPF in diabetic
rats. Further studies exploring in more detail the NO pathway and in particular
the use of more specific inhibitors of the various NO synthases are required
to delineate more accurately the role of NO in DN.
Extracellular Matrix Accumulation
A major feature of diabetic complications is extracellular matrix (ECM)
accumulation. Although ECM was originally viewed to be essentially inert,
it has now been shown not only to have a structural role but also to be in
a dynamic interaction with the surrounding cell population as well as being a
reservoir for various cytokines and growth factors. ECM consists of structural
proteins such as type IV collagen, cell-associated adhesion molecules such
as the integrins, antiadhesins and growth factors. The role of all these proteins

and their interactions in the genesis and progression of diabetic complications
is now an area of intensive investigation. There are major changes in gene
and protein expression of various ECM components in various sites of dia-
131Mechanisms of Diabetic Complications
betic vascular injury including activation of a range of growth factors such
as the prosclerotic cytokine, TGF- and matrix proteins such as type IV
collagen. Of particular interest are recent findings that various interventions
such as inhibition of advanced glycation and the RAS can influence expres-
sion of ECM components possibly via effects on cytokines such as TGF-
(fig. 1).
Transforming Growth Factor-
Transforming growth factor- (TGF-) has been shown to play a pivotal
role in ECM accumulation in the diabetic kidney. Renal expression of this
cytokine has been reported to be increased in both experimental and human
diabetes. Administration of antibodies to TGF- prevents diabetes-associated
renal hypertrophy. The role of this growth factor is further suggested by both
in vitro and in vivo findings indicating that putative mediators of DN such
as AII and AGEs promote expression of this cytokine. AII has been shown
in vitro to promote collagen IV production via TGF- in mesangial cells. In
the model of subtotal nephrectomy, an animal model of progressive renal
injury with many hemodynamic and structural similarities to diabetes, it has
been shown that in vivo inhibition ofthe actionof AII,either by ACE inhibition
or by AII receptor antagonism, is associated with reduced gene expression of
TGF-
1
. These treatments lead not only to reduced ECM accumulation but
attenuation of glomerular and tubulointerstitial injury and preservation of
renal function. More recently, a similar phenomenon has been observed in
experimental diabetes with reduced TGF-
1

gene expression after ACE inhibi-
tion, particularly in the tubulointerstitium. Reduced TGF-
1
expression after
ACE inhibition has also been observed in vessels from diabetic rodents.
Exogenous administration of AGEs upregulates a range of cytokines
including TGF- in the kidney. Recent studies have explored the relationship
between TGF-
1
, collagen and AGEs in diabetic vessels and shown that the
increase in gene expression of TGF-
1
in diabetic vessels can be prevented by
administration of the inhibitor of advanced glycation, aminoguanidine. Recent
studies suggest that these effects of inhibitors of glycation on growth factor
and structural protein expression are also observed in the diabetic kidney. At
this stage, no specific inhibitors of cytokine formation or action are available
for clinical use. Therefore, the major strategy for preventing renal TGF-
overexpression is via inhibition of stimuli of secretion of this prosclerotic
cytokine (fig. 1).
132Cooper