Pathogenesis of the glomerular abnormality in cyanotic congenital heart disease

Pathogenesis of the Glomerular Abnormality in Cyanotic Congenital Heart Disease Joseph K. Perloff,

MD,

Harrison Latta,

MD,

and Paola Barsotti,

PhD

We present evidence of 2 distinct glomerular abnormalities in cyanotic congenital heart disease—vascular and nonvascular— each believed to reflect a distinct pathogenesis. Glomeruli from both kidneys were studied with light microscopy in 13 necropsied cyanotic patients and in 8 controls. The vascular study characterized hilar arteriolar dilatation, capillary diameter, glomerular diameter, and capillary engorgement with red blood cells. The nonvascular study characterized juxtaglomerular cellularity, mesangeal cellularity, mesangeal matrix, focal interstitial fibrosis, and megakaryocytic nuclei per

cm2 of renal cortex. There was a significant increase in each of the above vascular and nonvascular items of interest relative to controls. Electron microscopy identified whole megakaryocytes with their cytoplasm in glomeruli. The vascular abnormality is believed to result from intraglomerular release of nitric oxide. The nonvascular abnormality is believed to result from plateletderived growth factor and transforming growth factor␤. 䊚2000 by Excerpta Medica, Inc. (Am J Cardiol 2000;86:1198 –1204)

ur previous renal studies in cyanotic congenital heart disease (CCHD) dealt with functional deO rangements. This study deals with morphologic de-

(mean 32). Hematocrit levels (Beckman/Coulter Inc., Brea, California) immediately before death were 55% to 75% (mean 61.4). No patient had cardiac or noncardiac disorders other than CCHD that might have caused renal pathology (Table 1). The interval between death and necropsy was 5 to 21 hours (mean 9.2 ⫾ 2.3). Diagnoses and causes of death are listed in Table 1. Data on glomerular vascular abnormalities included hilar arteriolar dilatation, capillary diameter, capillary engorgement with red blood cells, and glomerular diameter. Data on nonvascular glomerular abnormalities included mesangial cellularity, mesangeal matrix, juxtaglomerular cellularity, focal deep interstitial fibrosis, and megakaryocytic nuclei per cm2 of renal cortex. A scale of 0 (no increase), 1⫹, 2⫹, and 3⫹ was used to grade each of the above items of interest ⫹f (focal) ⫽ 0.5. Grading was done by the pathologist HL who was blinded to patients versus controls. All observations were converted into numbers, and t tests were used to test for differences. Glomerular capillary diameters were determined relative to diameters of red blood cells contained therein (Figure 1). The use of red cell diameter as a basis for estimating glomerular capillary diameter was justified by the absence of a significant difference between the mean of controls, 6.14 ␮m (SD ⫽ 0.84, n ⫽ 40) and the mean of patients, 6.09 ␮m (SD ⫽ 0.89, n ⫽ 50). Relatively small red cell size was caused by formalin fixation and processing. To determine whether there were significant diameter variations, 30 intraglomerular red cells were measured in 5 controls, in 5 patients, and in an intrarenal artery or vein lying in the plane of the light microscopy section. Glomerular diameters were determined as a fraction of the field with a 25⫻ objective, and were measured as the longest distance from the basement membrane of Bowman’s capsule on 1 side, to the basement membrane on the opposite side (Figure 1A). The width of

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rangements and their pathogenesis. In an investigation of renal pathology in 458 patients with congenital heart disease, the most striking abnormalities were in the 288 cyanotic patients whose glomeruli exhibited enlargement, dilatation of capillaries and hilar arterioles, red blood cell engorgement, hypercellularity, prominence of the juxtaglomerular apparatus, and prominence of the mesangium.3 However, distinct categories of morphologic abnormalities were not recognized. We identified 2 distinct histologic glomerular abnormalities: (1) vascular, represented by dilatation of the glomerular vascular bed; and (2) nonvascular, represented by an increase in juxtaglomerular cellularity and an increase in mesangial cells and mesangeal matrix.

METHODS

Light microscopy: Glomeruli from both kidneys were examined in 10 controls and in 13 patients with CCHD. The controls were devoid of heart disease, diabetes, hypertension, or any other disorder that might have had an impact on the kidneys. Five controls were men and 5 were women, aged 26 to 56 years (mean 38). Hematocrit levels immediately before death were 34% to 44% (mean 39). Seven patients were men and 6 were women, aged 19 to 58 years

From the Departments of Medicine, Pediatrics, Pathology and Laboratory Medicine, and the Ahmanson Adult Congenital Heart Disease Center, University of California at Los Angeles, Los Angeles, California; and Dipartimento di Medicina Sperimentale Patologia, Universita´ degli Studi di Roma, Rome, Italy. Manuscript received April 5, 2000; revised manuscript received and accepted June 9, 2000. Address for reprints: Joseph K. Perloff, MD, Division of Cardiology, Room 47-123, UCLA Center for the Health Sciences, 10833 LeConte Avenue, Los Angeles, California 90095-1679. E-mail: jperloff@ mednet.ucla.edu.

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©2000 by Excerpta Medica, Inc. All rights reserved. The American Journal of Cardiology Vol. 86 December 1, 2000

0002-9149/00/$–see front matter PII S0002-9149(00)01202-9

TABLE 1 Cyanotic Congenital Heart Disease Congenital Malformation Eisenmenger syndrome Ventricular septal defect Ventricular septal defect Ventricular septal defect Ventricular septal defect Aortopulmonary window Univentricular heart Univentricular heart Univentricular heart Atrioventricular septal defect Fallot’s tetralogy pulmonary atresia, Potts, pulmonary vascular disease Tricuspid atresia, pulmonary stenosis, Potts, pulmonary vascular disease Shone syndrome, patent ductus arteriosus, pulmonary vascular disease Complete transposition of great arteries, ventricular septal defect, pulmonary vascular disease

Cause of Death Sudden, unexplained Ascending aortic dissection Sudden, unexplained Vasopastic cerebral infarct (migraine) Atrial fibrillation ventricular failure, cardiac arrest Sudden, unexplained Atrial fibrillation, ventricular failure Cardiac arrest Sudden, unexplained Right ventricular failure Left ventricular failure

Atrial fibrillation, biventricular failure Cardiac arrest

the microscopic field was 620 ␮m, a measurement that permitted the mean and SD to be converted into micrometers. Megakaryocytes were identified by characteristic large dark-staining multilobulated nuclei (Figure 2) whose shape tended to be altered by wedging in glomerular capillaries. Megakaryocytic cytoplasm could not be identified by light microscopy. Electron microscopy: Tissue was immediately fixed in 2.5% glutaraldehyde and 2% paraformaldehyde, then postfixed with 2% osmium tetroxide stained with uranyl acetate and lead citrate and embedded in an epoxy resin. Ultrathin sections were examined with a Zeiss 109 electron microscope (Carl Zeiss, Thornwood, New York). Megakaryocytes were identified by characteristic multilobulated nuclei, demarcating membrane systems, and cytoplasmic granules, and were classified according to stage of maturity (Figures 3 and 4).4 Glomeruli were examined by electron microscopy in fresh necropsy sections from 3 cyanotic patients, aged 19, 24, and 31 years, from renal biopsy specimens in 3 patients (2 aged 15 months and 16 years with CCHD who did not undergo operation, and 1 aged 14 years 4 weeks after cardiac surgery had eliminated the cyanosis). The 3 necropsy specimens were also examined by light microscopy for glomerular megakaryocytic nuclei per cm2 renal cortex. Electron microscopy was used for 2 purposes: to examine the structure of the mesangium, its cellular and extracellular components, and its relation to adjacent structures (Figure 5)5; and to identify megakaryocytic cytoplasm within glomeruli.

RESULTS

Light microscopy: (Table 2) Dilated hilar arterioles (Figures 1B and 6A): Hilar arterioles were 1⫹ to 3⫹

dilated in patients relative to controls (p ⬍0.001). Glomerular capillary diameter: Mean diameter in controls was 9.05 ␮m (SD 2.26, n ⫽ 75), and in patients was 12.88 ␮m (SD 3.52, n ⫽ 75). The difference was highly significant (p ⬍0.001, 2 tailed). Red blood cell engorgement of glomerular capillaries: Two controls had scattered focal engorgement (⫹f). Eight patients had 2⫹ to 3⫹ generalized engorgement (p ⬍0.01). Glomerular diameter (Figure 1B): Diameter in controls was ⱕ0.3 of the field with a 25x objective, and in 11 patients was ⬎0.3 (p ⬍0.01). When converted into micrometers, mean diameter in controls was 170 ⫾ 15.5, and in patients was 276.5 ⫾ 25.1 (p ⬍0.01). Juxtaglomerular cellularity: Two controls had a 1⫹ focal increase. Thirteen patients had 1⫹ to 3⫹ generalized increases, p ⬍0.001. Mesangial cells (Figures 1B and 1C): Two controls had scattered focal increases (⫹f). Ten patients had 1⫹ to 3⫹ increases (p ⬍0.001). Mesangial matrix (Figure 6): Two controls had scattered focal increases (⫹f). Eleven patients had 1⫹ to 3⫹ increases (Figures 1B and 1C and 6). There was a high correlation between the increase in mesangeal matrix and the increase in mesangeal cells (r ⫽ 0.82, p ⬍0.0006). Deep focal cortical lesions: Seven patients had unusual deep focal lesions ⱕ1 mm in diameter distributed throughout the cortex. These lesions consisted of hyalinized glomeruli, atrophic tubules, and interstitial fibrosis. Controls had no such lesions. Megakaryocytic nuclei (Figure 2): The number of nuclei per cm2 of renal cortex in controls was 0 to 1.4 (mean 0.4). The number in patients was 3.6 to 41.4 (mean 10.7) (p ⬍0.001). Electron microscopy (Figures 3 and 4): Characteristic multilobulated megakaryocytic nuclei with intact cytoplasm, demarcating membrane systems, cytoplasmic granules, and clusters of platelets were identified in glomerular capillaries of 5 cyanotic patients studied. The number of megakaryocytic nuclei per cm2 of renal cortex in the 3 necropsy specimens examined by light microscopy was 8.6 to 12.8 (mean 10.3), and in the controls 0 to 1.4 (mean 0.4). Megakaryocytes were not found in the 1 renal biopsy specimen obtained after cardiac surgery had eliminated the cyanosis.

DISCUSSION

Vascular glomerular abnormalities: The erythrocytosis of CCHD is accompanied by an increase in whole blood viscosity1 and an increase in intraglomerular endothelial shear stress.6 – 8 Ultrafiltration augments the high viscosity inherent in erythrocytosis, disproportionately increasing glomerular vascular resistance and shear stress. Nitric oxide (NO) is synthesized by NO synthase in glomerular mesangial cells, capillary and juxtamesangial endothelial cells, and in specialized cells of the macula densa (Figure 5),6 –9 and acts as an autocrine and paracrine hormone that governs the glomerular vascular response to endothelial shear stress.7,8,10 Mesangial cells proliferate in response to platelet-derived growth factor (PDGF),11 potentially increasing the substrate for intrarenal release of NO (see next section). We propose that the increase in shear stress of the erythrocytotic glomer-

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FIGURE 1. A, control glomerulus (light microscopy) with its delicate capillary network (CN) lined by a thin layer of endothelial cells, a central region of mesangial cells surrounded by mesangial matrix (MM), Bowman’s space (BS) between the 2 epithelial layers, and a delicate Bowman’s capsule (BC). Periodic acid Schiff stain ⴛ210. B, enlarged glomerulus from a cyanotic patient with striking dilatation of the afferent hilar arteriole (curved white arrows) and its immediate extension into the glomerulus, and an increase in mesangeal cellularity. Bowman’s space is virtually filled by the enlarged vascular glomerulus. Hematoxylin-eosin stain ⴛ180. C, an enlarged glomerulus from a cyanotic patient showing dilatation of glomerular capillaries (smaller oblique arrows) in addition to an increase in mesangeal cellularity. Large central arrow identifies a megakaryocytic nucleus. Hematoxylin-eosin stain ⴛ180.

ular perfusate excites release of NO,6,8,10 in response to which the glomerular vascular bed dilates (Figures 1B and 1C, and 6A). The dilated, enlarged, engorged glomeruli may double glomerular mass and renal cortical vascularity.3 That the glomerular vascular abnormality— dilatation— coincides with an increase in red cell mass is supported by histologically similar responses to experimental hypoxemia,12 high altitude hypoxemia,13 and normoxemic erythropoietin-induced erythrocytosis.10 The common denominator appears to be erythrocytosis. Nonvascular glomerular abnormalities: We postulate that the glomerular cellular and connective tissue abnormalities are responses to PDGF and transforming growth factor-␤ (TGF-␤) carried to glomeruli in cytoplasmic granules of intact megakaryocytes that enter the systemic arterial circulation via right-to-left shunts14 –20 (Figures 2 to 4). The idea that these mitogens and cytokines play a role in glomerular disease is 1200 THE AMERICAN JOURNAL OF CARDIOLOGY姞

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not new.21–23 Our hypothesis is based on the following observations and lines of reasoning. In proposing a pathogenetic link between PDGF/ TGF-␤ and the nonvascular glomerular abnormalities in CCHD, 2 steps were necessary: The first step— without which the second could not follow—was a major focus of this investigation, namely, identification of a vehicle for delivery of the inciting cytokines and mitogens to the glomeruli. The second step—the subject of a separate study—is identification of intraglomerular PDGF and TGF-␤ using in situ hybridization and immunohistochemistry.24,25 The first step was more essential to our argument because mesangial cells and glomerular endothelial cells express a PDGF-like protein,13 so that the presence of intraglomerular PDGF, per se, would not necessarily reflect its source, which we sought to identify. There is compelling evidence that whole megakaryocytes pass through bone marrow sinusoids, enter DECEMBER 1, 2000

FIGURE 2. Glomeruli from 2 cyanotic patients. Arrows point to typical large dark-staining multilobulated megakaryocytic nuclei. Hematoxylin-eosin stain (left ⴛ450; right ⴛ650, reduced by 24%).

FIGURE 3. Renal biopsy from a cyanotic patient, examined with transmission electron microscopy (ⴛ32,000, reduced 25%). A mature intraglomerular megakaryocyte with multilobulated nucleus (N) is seen. The cytoplasm is divided into platelet fields by an extensive demarcating membrane system (DMS). CG ⴝ cytoplasmic granules.

the systemic venous bed, circulate as normal constituents, and shed platelets from their cytoplasm during passage through the lungs14 –20,26 (see below). It has been argued persuasively that right-to-left shunts deliver whole megakaryocytes from the systemic venous to the systemic arterial circulation where they may lodge in capillaries of the digits and periosteum, release PDGF and TGF-␤, and cause clubbing and hypertrophic osteoarthropathy.27,28 Megakaryocytic nuclei have been found at necropsy in clubbed fingers of cyanotic patients.29 We present evidence that the nonvascular glomerular abnormality in CCHD may result from locally released PDGF and TGF-␤ in cytoplasmic granules of systemic venous megakaryocytes that are shunted into the systemic arterial circulation with their cytoplasm and fortuitously impact in glomerular

capillaries. Importantly, in patent ductus arteriosus with pulmonary vascular disease and reversed shunt, the toes are clubbed but the fingers are not, an observation consistent with the proposal that a formed element in the systemic venous circulation selectively finds its way into the systemic arterial circulation distal to the left subclavian artery. PDGF is a potent mitogen that binds with high affinity to responsive cells, exerts its action locally because of an extremely short half-life, is a growth factor for mesenchymally derived cells, and promotes protein synthesis, connective tissue formation, and cellular proliferation.13 TGF-␤ is a cytokine that regulates tissue repair, is chemotactic for neutrophils, T cells, monocytes, and fibroblasts, and enhances deposition of extracellular matrix.22,23 We propose that the

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FIGURE 4. Renal biopsy from a cyanotic patient, examined with transmission electron microscopy (ⴛ8,900, reduced 28%). A mature intraglomerular megakaryocyte contains well-formed platelets (PL) with cytoplasmic granules (CG). RBC ⴝ red blood cell. N ⴝ nucleus.

FIGURE 5. Diagram of the glomerular mesangial region based on transmission electron microscopy. The mesangium is at the center of capillaries (c) that form a glomerular lobule. Portions of mesangium are covered by the lamina densa (LD) and lamina rara externa (LRE). Mesangial cells (M) are adjacent to glomerular capillaries. Fenestrations (F) penetrate central portions of endothelial cells and allow plasma to enter intercellular mesangeal channels (IC). En ⴝ endothelium; Ep ⴝ epithelium; LRI ⴝ lamina rara interna; other abbreviations as in Figures 1 and 4. (Adapted from Latta H.5)

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observed increases in mesangial cells, mesangeal matrix, and juxtaglomerular cellularity may be responses to PDGF and TGF-␤ carried in the cytoplasm of shunted systemic venous megakaryocytes, and released within glomerular tufts. Because NO is antiproliferative,6 the increase in glomerular cellularity is not likely to represent a response to NO (see earlier). The validity of our hypothesis regarding the pathogenesis of the nonvascular glomerular abnormality depended on evidence from 2 sources. First, light microscopy determined that the number of megakaryocytic nuclei per cm2 of renal cortex in CCHD significantly exceeded the number of megakaryocytic nuclei in control glomeruli. Second, electron microscopy established the presence of whole megakaryocytes with their cytoplasm within glomeruli. Crucial to our argument is that whole megakaryocytes leave bone marrow sinusoids, circulate in the systemic venous bed, and divest their cytoplasm during the pulmonary transit, in the process of which platelets are formed.15–20 The following points are consistent with this argument: The number of platelets in the pulmonary venous circulation appreciably exceeds the number in the pulmonary arterial circulation.17 A single megakaryocyte, depending on its ploidy, produces approximately 4,000 to 8,000 platelets, and an estimated 40,000 megakaryocytes are delivered to the pulmonary circulation per minute.19 These estimates coincide with the number of platelets in pulmonary veins com-

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TABLE 2 Cyanotic Congenital Heart Disease Patients Vascular Observations Dilated Hilar Arterioles

Engorgement of Glomerular Capillaries With RBCs

Glomerular Capillary Diameter/RBC Diameter

Nonvascular Observations

Glomerular Diameter

Mesangial Cells

Mesangial Matrix

Juxtaglomerular Cellularity

Deep Focal Cortical Lesions

Megakaryocytic nuclei/cm2 Renal Cortex

⫹ ⫹ ⫹⫹⫹ 0 ⫹ ⫹⫹⫹ 0 ⫹ ⫹ 0 ⫹⫹⫹ ⫹⫹ ⫹

⫹⫹ ⫹⫹ ⫹⫹ ⫹ ⫹⫹⫹ ⫹⫹ ⫹⫹⫹ ⫹ ⫹ ⫹⫹⫹ ⫹⫹ ⫹ ⫹⫹

0 0 ⫹ 0 0 ⫹ ⫹ ⫹ 0 ⫹ ⫹ 0 ⫹

4.4 5.4 6.3 18.2 41.4 5.2 11.8 3.6 9.2 5.8 4.2 6.3 8.7

⫹focal ⫹focal 0 0 0 0 0 0

⫹focal ⫹focal 0 0 0 0 0 0

0 0 0 0 0 0 0 0

0.4 0.4 0 0.3 1.4 0 0.6 0

Patients ⫹⫹⫹ 0 ⫹⫹⫹ ⫹ ⫹⫹⫹ ⫹⫹ ⫹ ⫹⫹ 0 ⫹ 0 ⫹⫹ ⫹⫹⫹

⫹⫹⫹ ⫹⫹ ⫹⫹ ⫹ ⫹⫹focal ⫹ 0 ⫹ 0 ⫹⫹ ⫹ ⫹⫹ ⫹⫹

2–3 2–3 2–3 2–3 3 2–3 1.5 2–3 1.5 2–3 1–2 2–4 3–5

0.5 0.5 0.5 0.4 0.5 0.5 0.4 0.5 0.3 0.4 0.6 0.6 0.5

⫹⫹⫹ ⫹ ⫹⫹ 0 ⫹ ⫹⫹⫹ ⫹ ⫹ ⫹ 0 ⫹⫹ ⫹ ⫹⫹ Controls

0 0 0 0 0 0 0 0

0 0 0 0 0 ⫹focal 0 ⫹focal

1.5 1.5 2 2 1 2 2 2

0.3 0.25 0.3 0.25 0.3 0.25 0.3 0.3

⫹focal ⫹focal 0 0 0 0 0 0

RBC ⫽ red blood cells. A scale of 0 ⫽ no increase; ⫹, ⫹⫹, ⫹⫹⫹ ⫽ grade for each of the above items of interest; ⫹ focal ⫽ 0.5.

FIGURE 6. A, enlarged glomerulus in a cyanotic patient. There is striking dilatation of the afferent arteriole (AA) with dilatation of its extension into the glomerulus (smaller paired arrows), in addition to an increase in mesangeal and juxtaglomerular cellularity and mesangial matrix (MM). Periodic acid-Schiff stain ⴛ220. B, glomerulus in a cyanotic patient showing an increase in mesangial matrix and thickening of Bowman’s capsule (BC). Vascular dilatation is inconspicuous, whereas mesangial matrix is prominent. Periodic acid-Schiff ⴛ220 (A and B, reduced by 30%).

pared with pulmonary arteries (see above).18 –20 When a megakaryocyte divests itself of cytoplasm during its pulmonary transit, the naked nucleus can traverse the pulmonary capillaries, escape phagocytosis, and enter the systemic arterial circulation.19 Megakaryocytic nuclei have been identified at necropsy in all lungs so studied, 62% of spleens and 36% of kidneys.30 The number of megakaryocytic nuclei per cm2 of renal cortex in our cyanotic patients significantly exceeded the number in the controls (Table 2) (p ⬍0.001),

supporting the conclusion that whole megakaryocytes with their cytoplasm were shunted from the systemic venous into the systemic arterial circulation and were delivered to glomerular tufts. Megakaryocytes with characteristic multilobulated nuclei and intact cytoplasm were identified by electron microscopy within glomerular capillaries of all cyanotic patients so studied (Figures 3 and 4). The significantly larger number of megakaryocytic nuclei in glomeruli of our cyanotic patients was evidence that

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intact megakaryocytes were delivered by the right-toleft shunts into the systemic arterial circulation and into glomerular tufts (Table 2 and Figure 2). The occasional megakaryocytic nuclei in glomeruli of our controls represented the anticipated small number of naked nuclei that traverse the pulmonary vascular bed, escape phagocytosis, enter the systemic arterial circulation, and impact glomerular tufts16,17 (Table 2). Despite the glomerular abnormalities described herein, renal function in CCHD typically remains normal1 with 2 exceptions. First, inappropriately low uric acid excretion results in hyperuricemia.2 Second, the glomerulus is normally porous to albumin, which is nevertheless retained because glomerular capillary walls are negatively charged, whereas the protein molecule is cationic.1 Erythrocytosis increases perfusion pressure, overcoming the cationic effect, so albumin leaves the glomerular circulation resulting in proteinuria.1 Conclusion: We identified 2 distinct histologic glomerular abnormalities in CCHD—vascular and nonvascular—and we proposed 2 distinct pathogenetic mechanisms. The vascular abnormality is characterized by dilated hilar arterioles, an increase in glomerular capillary diameter with red cell engorgement, and glomerular enlargement for which the following pathogenetic mechanism was proposed. Erythrocytosis is accompanied by an increase in whole blood viscosity that is augmented by ultrafiltration. The high viscosity is accompanied by an increase in endothelial shear stress, stimulating intraglomerular release of NO in response to which the glomerular vascular bed dilates, resulting in the observed vascular abnormality. The nonvascular glomerular abnormality is characterized by an increase in juxtaglomerular and mesangeal cellularity and an increase in mesangeal matrix for which the following pathogenetic mechanism was proposed. Megakaryocytic cytoplasm contains high concentrations of PDGF and TGF-␤ that exert their actions locally. Whole megakaryocytes with intact cytoplasm leave bone marrow sinusoids, circulate in the systemic venous bed, enter the systemic arterial bed via right-to-left shunts, fortuitously lodge in glomeruli, and release their mitogens and cytokines that are responsible for the observed nonvascular abnormality. Acknowledgment: We are pleased to acknowledge Alan Garfinkel, PhD, Associate Professor of Medicine and Physiology, for critically reviewing the statistics.

1. Perloff JK. Cyanotic congenital heart disease is a multisystem systemic disorder. Exp Clin Cardiol 1999;4:77–79.

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2. Ross EA, Perloff JK, Danovitch GM, Child JS, Canobbio MM. Renal function and urate metabolism in late survivors with cyanotic congenital heart disease. Circulation 1986;73:396 – 400. 3. Spear GS. Glomerular alterations in cyanotic congenital heart disease. Bull Johns Hopkins 1960;106:347–367. 4. Fawcett DW, Raviola E. Bone marrow and blood cell formation. In: Bloom W, Fawcett DW, eds. A Textbook of Histology, 12th ed. New York: Chapman and Hall, 1994:234 –259. 5. Latta H. An approach to the structure and function of the glomerular mesangium. J Am Soc Nephrol 1992;2:S65–S73. 6. Raij L, Shultz PJ. Endothelium-derived relaxing factor, nitric oxide: effects on and production by mesangial cells and the glomerulus. J Am Soc Nephrol 1993;3:1435–1441. 7. Wilcox CS, Welch WJ, Murad F, Gross SS, Taylor G, Levi R, Schmidt HHW. Nitric oxide synthase in macula densa regulates glomerular capillary pressure. Proc Natl Acad Sci 1992;89:11993–11997. 8. Imig JD, Roman RJ. Nitric oxide modulates vascular tone in preglomerular arterioles. Hypertension 1992;19:770 –774. 9. Mene P, Simonson MS, Dunn MJ. Physiology of the mesangial cell. Physiol Rev 1989;69:1347–1411. 10. Wilcox CS, Deng X, Doll AH, Snellen H, Welch WJ. Nitric oxide mediates renal vasodilatation during erythropoietin-induced polycythemia. Kidney Int 1993;44:430 – 435. 11. Schultz PJ, Di Corletto PE, Silver BJ, Abooud HE. Mesangial cells express PDGF mRNAs and proliferate in response to PDGF. Am J Physiol 1988;255: F674 –F684. 12. Spear GS, Kihara I. The glomerulus and serum sickness in experimental hypoxia. Br J Exper Pathol 1972;53:265–276. 13. Naeye RL. Children at high altitude: pulmonary and renal abnormalities. Circ Res 1965;16:33–38. 14. Hansen M, Pedersen NT. Circulating megakaryocytes in blood from the antecubital vein in healthy adult humans. Scand J Haematol 1978;20:371–376. 15. Trowbridge EA, Martin JF, Slater DN. Evidence for a theory of physical fragmentation of megakaryocytes, implying that all platelets are produced in the pulmonary circulation. Thromb Res 1982;28:461– 475. 16. Kaufman RM, Airo R, Pollack S, Crosby WH. Circulating megakaryocytes and platelet release in the lung. Blood 1965;26:720 –731. 17. Becker RP, DeBruyn PPH. The transmural passage of blood cells into myeloid sinusoids and the entry of platelets into sinusoidal circulation. Am J Anat 1976;145:183–205. 18. Tinggaard-Pedersen N. Occurrence of megakaryocytes in various vessels and their retention in the pulmonary capillaries of man. Scand J Haematol 1978;21: 369 –375. 19. Tavassoli M. Megakaryocyte-platelet axis and the process of platelet formation and release. Blood 1980;55:537–545. 20. Slater DN, Trowbridge EA, Martin JF. The megakaryocyte in thrombocytopenia: a microscopic study which supports the theory that platelets are produced in the lungs. Thromb Res 1983;31:163–176. 21. Remuzzi G. Role of platelets in progressive glomerular disease. Pediatr Nephrol 1995;9:495–502. 22. Sharma K, Ziyadeh FN. The emerging role of transforming growth factor ␤ in kidney diseases. Am J Physiol 1994;266:F829 –F842. 23. Zoja C. Expression of transforming growth factor ␤ isoforms in human glomerular diseases. Kidney Int 1996;49:461– 469. 24. Yoshimura A, Gordon K, Alpers CE, Flaege J, Pritzl P, Ross R, Couser WG, Bowen-Pope DF, Johnson RJ. Demonstration of PDGF ␤ chain mRNA in glomeruli in mesangial proliferative nephritis by in situ hybridization. Kidney Int 1991;40:470 – 476. 25. Gesualdo L, DiPaolo S, Milani S, Pinzani M, Grappone C, Ranieri E, Pannarale G, Schena FD. Expression of platelet-derived growth factor receptors in normal and diseased human kidney. An immunohistochemistry and in situ hybridization study. J Clin Invest 1994;94:50 –58. 26. Tavassoli M, Aoki M. Migration of entire megakaryocytes through the marrow-blood barrier. Br J Haematol 1981;48:25–29. 27. Martinez-Lavin M. Pathogenesis of hypertrophic osteoarthropathy. Clin Exper Rheum 1992;10(suppl 7):49 –50. 28. Dickinson CJ. The etiology of clubbing and hypertrophic osteoarthropathy. Eur J Clin Invest 1993;23:330 –338. 29. Fox SB, Day CA, Gatter KC. Association between platelet microthrombi and finger clubbing. Lancet 1991;338:313–314. 30. Brill R, Halpern MM. The frequency of megakaryocytes in autopsy sections. Blood 1948;3:286 –291.

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