Dr. H. Novadian, SpPD K-GH FINASIMDiv. Nephrology and Hypertension. Dept. of MedicineMedical Faculty, University of Sriwijaya Dr. Moh Hosein Hospital, Palembang

J.G.App.Afferent.ADCT Efferent.ACap. loopsDCT

1. Fenestrated Endothelium2. Lamina Rara Interna3. Lamina Rara Densa4. Lamina Rara Externa5. Podocytes + Slit membraneCapillary Lumen

Glomerular Capillary LumenBowmans Capsule SpaceBLOOD

Proteins3.6nm/70,000MWPlasma - Proteins

FILTRATE

glomerulonephritis- etiology -primary (idiopathic)- pathology is confined to the kidney- any systemic features as direct consequence of glomerular dysfunction

secondary- kidney abnormality as a part of multi- system disorder

Glomerular diseases:

Primary GN: Acute Diffuse Prol. Poststrept & other Inf. Crescentic (Rapidly Progressive) Membranous GN. Lipoid / Minimal Change . Focal segmental G.sclerosis. Membranoproliferative GN. IgA nephropathy. Chronic Idiopathic GN.Associated with Systemic Diseases: SLE, DiabetesMellitus. Goodpasture's Syndrome. Polyarteritis Nodosa. Wegener's Granulomatosis. Henoch-Schonlein Purpura. Bacterial Endocarditis. Amyloidosis Hereditary Disorders:Alport's Syndrome.Fabry's Disease

Primary renal diseaseNephritis Post Infectious IgA nephropathy RPGN- Anti GBM- IdiopathicNephrotic SyndromeMinimal Change Focal Sclerosis Membranous nephropathy Membranoproliferative GN Systemic disease Vasculitis - SLE - HSP- PAN - EMC- WagenersDiabetes Amyloid

glomerulonephritis- chronology -acute:days to weeks

subacute/rapidly progressive:over weeks to few months

chronic:many months to years

glomerulonephritis- location -focal:

Glomerular damage - patterns:

glomerulonephritis- pathology -membranous:- expansion of glomerular basement membrane as a dominant feature

sclerosis:- increased amount of homogenous non-fibrillar extracellular material (similar to GBM and mesangeal matrix)

fibrosis- deposition of type I and III collagen- commonly as a consequence of healing of crescents or tubulointerstitial inflammation

PathogenesisPrimary insultmajor insult to glomerulusa. immune attackb. metabolic stressc.mechanical stress

1. Immune GlomerulonephritisIn-Situ immune complex formation:Tissue antigens - Goodpasture anti GBM AgPlanted antigens - infections, toxins, drugs.Circulating immune complex deposition.Endogenous - DNA as in SLEExogenous - infections.Cell mediated Immune injury

Immune Glomerulonephritis:C.Immune Complex ANTI-GBM HEYMANN

Pathogenesis of Immune GN:Ab, Ag/Ab or Immune complex deposition.Immune reactionInflammation Activation of complementdestruction of glomerular structureRenal dysfunction, Proteinuria, Hematuria

Immune Glomerulonephritis:

2. Metabolic stress (metabolic injury)Hyperglycemia ( Diabetes Melitus)(1)advanced glycosylation end-products (AGEs)(2)reactive oxygen speciescell sorbitol accumulationmitogen-activated protein kinase activation(3)high glucose-triggered glomerular hypertension

(i)mesangial cell hypertrophy(ii)increased mesangial cell matrix production(iii)reduced matrix catabolism(iv)glomerulosclerosis

3. Mechanical stress(hemodynamic glomerular injury)systemic hypertensionmalignant hypertensionmassive fibrinoid necrosis of afferent arteriole and glomerulithrombotic microangiopathy

nephritic urinary sedimentacute renal failurechronic sustained hypertensionarteriolar vasoconstriction and sclerosis

secondary glomerular and tubulointerstitial atrophy and sclerosis

mechanical stress(hemodynamic glomerular injury)glomerular hypertensionglomerular hypertension as an adaptive response to increased workload 2 to loss of other nephrons

sustained glomerular hypertension

increased mesangial matrix production

glomerulosclerosis

Phatophysiology of Diabetic Nephropathy

Glomerulosclerosis

Glomerular capillary hypertensionGlomerular pressure injuryOxidative stress

Chronic kidney disease

Cell and tissue growth

Inflammation

Angiotensin II

Brewster, Perazella. Am J Med 2004;116:263272.

Reduction in nephron mass

TGF-TGF- plays a key role in extracellular matrix formation in mesangiumand interstitium that leadsto fibrosis and loss of nephron unitswww.hypertensiononline.org

bFGFPDGFAng IITSP1TGF-O2TGF- plays a key role in extracellular matrix formation in mesangiumand interstitium that leadsto fibrosis and loss of nephron unitswww.hypertensiononline.org

TIMPbFGFPDGFAng IIProteases(-)(-)(+)(+)(+)TSP1ET-1PAI-1O2TGF-TGF- plays a key role in extracellular matrix formation in mesangiumand interstitium that leadsto fibrosis and loss of nephron unitswww.hypertensiononline.org

Nephritic Syndromes :Diffuse Proliferative GNPost Streptococcal.Rapidly Progressive GN (or Crescentic)Post Streptococcal, Goodpastures, Focal GlomerulonephritisPrimary: Bergers disease (IgA Nephritis)Secondary IgA nephritis, Henoch Schonlein purpura, SBE, Coeliac Disease etc.

acute nephritic syndrome

Oliguria Abnormal of Urinalysis (proteinuria >, hepatomegali) edema.

The Ethiology of ANSGlomerulopathy (GP) idiopatikGP acute proliferativeGP mesangioproliferatif nefritis Ig A or Berger diseaseGP membrnoproliferativeGlomerulopathy postinfectionPost streptococcus beta hemoliticusBacterialis EndocarditisStaphylococcus albus (shut nefritis)Viseral AbscesHepatitis B antigenemiaDisseminated Lupus Erythematosus (DLE)Vasculitis Poliarteritis nodosaGranulomatosis WagenerHenoch-Schonlein Purpura (HSP)Nefritis herediter

Pathogenesis and patophysiology of ANS

1. Kelainan urinalisis: kerusakan dinding kapiler glomerulus 2. Penurunan LFG: penurunan filtrasi bagian distal mempertahankan reabsorbsi Na dan air retensi Na dan air cairan ektra seluler 3. Hipotesis bendungan paru akut : 1. Vaskulitis umum 2. Penyakit jantung hipertensi 3. Miokarditis 4. Hipervolome dan retensi Na

Diagnosis klinis: - Foto dada : Kardiomegali,bendungan paru- EKG: Voltase rendah,T inverted,QT panjang- Kelainan urinalisis : Proteinuria, hematuria

Diagnosis perjalanan penyakit (komplikasi)- Faal ginjal : kenaikan ureum & creatinin- Elektrolit :hiponatremi dilusi,hiperkalemi- Serum protein & profil lipid : hipoalbuminemia- Faktor pembekuan : kenaikan faktor pembekuan.

Pemeriksaan Imunodiagnosis

1. ANA : LES2. Faktor rematoid: penyakit infeksi kronis3. Ag hepatitis B: GN membraneus anak 4. Krioglobulin: CIC5. Komplemen serum: C1q,C4,C3 menurun Nefropati IgA6. CICx 7. Histopatologi ginjal : pem imunofluoresen

A.Pengobatan Darurat medis SNA1.Bendungan sirkulasi dan paru furosemide 40-80mg IV morfin (bila tersedia) obat anti hipertensi oral Dialisis : GGA 24jam konservatif gagal2.Ensefalopati hipertensif akut Hidralazin 20 mg IV dgn furosemid Nifedipin IM/SL dgn furosemid

B.SuportifDiet: 35kal/KgBB/hr,lemak tdk jenuh,prot hewani

Cairan: dibatasi untuk keseimbangan cairan tubuh.Lanjutan

Poststreptococcal GNUsually occurs 10 days after pharyngitis and 14 days after skin infection (not synpharyngitic)Fallen incidence in US, but common in some rural areas, poor hygiene places, and tropical countriesOccurs more often in males and children

Post Streptococcal GN (Prol.GN):1-4 weeks following streptococcal infection (nephritogenic strains)Immune mediated (time for Ab formation)Granular deposits of IgG,IgM & C3 in GBM, (subepithelial location common)Humps in GBM on EM or IF Microscopy

Poststreptoccal GN (nephritic strains)

Known nephritic strains include M types 1, 2, 4, 12, 18, 25, 49, 55, 57, 60Many proposed mechanisms: Molecular mimickry vs. autoimmune vs. polyclonal activation of B lymphocytesRepeat infections are not common as immunity is type specific and not usually transient

Clinical PresentationMost patients have milder diseaseClassically, presents with overt nephritic syndrome and oliguric ARFSymptoms can include gross hematuria (100% microscopic), HA, htn (60-80%), hypervolemia, and edema (80-90%)

Clinical PresentationNephritic urinary sediment dysmorphic RBCs, red cell casts, leukocytes, subnephrotic proteinuriaNephrotic-range proteinuria not common

Clinical Features: G.NephritisHypertensionSkin InfectionsCongestive Cardiac Failure

Labs

Serum Cr can be commonly elevated at presentation, though mildC3 and CH50 decreased w/in 2 weeksC4 usually normal (complement level usually normal within 6-8 weeks)Most patients have directed Ab, such as ASO, anti-DNAse B, etcSerum IgG and IgM increased in 80% and returns to normal in 1-2 monthsPolyclonal cryoglobulinemia in 75%

Laboratory Features: G.NephritisInflammationDecreased filtrationDamage to filtration unit

poststreptococcal glomerulonephritisTx:(1) antibiotics to eliminate streptococcal infection(2) supportive therapy until spontaneous resolution(3) diuretics(4) antihypertensive agent(s)(5) dialysis rarely needed (to control hypervolemia or uremic syndrome)prognosis:excellentspontaneous resolution in childrensome degree of persistent proteinuria in 20% of adults

PengobatanSimptomatis mencegah penyulit fatal: 1. Istirahat: fase akut tak lebih 3 bulan 2. Diet selama fase oliguria/anuria: protein dibatasi 0,5-0,75 gram/kgbb protein hewani nilai biologis tinggi KH: 35 cal/kgbb, Lemak tidak jenuh Elektrolit: Na dibatasi, K( 70-90meq/hari)3.Antibiotika PP 2x 600.000 IU 7 hari oral 2x 200.000iu fase konvalesen

Post Streptococcal GN Course Acute Renal failure : 5% Chronic Renal Failure : 2.5 %

Sign

Diuresis HypertensionCr to Normal Em Humps Hematuria Proteinuria Resolution

1 week 2 week 3-4 week 6-7 week 3-6 week 3 year

Prognosis

GNA pasca streptokok anak : baikDewasa: penyembuhan (80-90%), meninggal fase akut (0,5 %), RPGN (5-10%), kronis (5-10%)Tanda prognosis buruk : oliguri/anuri beberapa minggu, penurunan LFG, hipokomplemenemia menetap, kenaikan kosentrasi circulating fibrinogen fibrin complexes dan kenaikan FDP dalam urin

acute nephritic syndrome and RPGN- etiology -immune-complex glomerulonephritisin situ formationglomerular trapping of circulating complex

idiopathicwith known antigenic stimulus (infection-associated GN)part of multisystem disorder

acute nephritic syndrome and RPGN- etiology -anti-GBM diseasesautoantibody directed at 28 kDa Ag on 3 chain of type VI collagen

pauci-immune glomerulonephritis (ANCA-associated small vessel vasculitis)idiopathic renal-limited crescentic glomerulonephritismicroscopic polyangiitis nodosaWegeners granulomatosis

immune complex GNpauci-immune GNanti-GBM diseaseserum complementanti-GBMANCAnormalnormalnegnegneg**neg**posposlow* *: exception IgA nephropathy (normal)**: ~20% have low level ANCA

acute nephritic syndrome and RPGNimmune-complex glomerulonephritisas a part of multisystem disorderLupus nephritisseen 40-85% of SLE patientstriggered by formation of immune complexes within glomerular capillary wallLab: range of serologic abnormalitieshypocomplementemia (75 to 90%)antinuclear Abs (ANA) (95 to 99%)anti-double-stranded DNA (ds-DNA) Abs

acute nephritic syndrome and RPGNimmune-complex glomerulonephritisLupus nephritislupus-related antiphospholipid antibody syndrome

tissue plasminogen activator2-antiplasmin

variable degree of renal impairmentintervascular microthrombiswelling of endothelial cellsthrombosis

acute nephritic syndrome and RPGNimmune-complex glomerulonephritisLupus nephritisTx:treatment is based largely on (1) class of injury and (2) disease activityrenal biopsy: important guide to therapynormal bxno therapymesangial deposit only

proliferative nephritisglucocorticoidscyclophosphamidemycophenolate mofetiltherapeutic option for cyclophosphamide resistant case

acute nephritic syndrome and RPGNimmune-complex glomerulonephritisas a part of multisystem disordercryoglobulinemia glomerulopathymixed cryoglobulinemias (types II & III)femalesixth decadevariable combination of leukocytoclastic vasculitisskin ulcerationarthralgiafatigueRaynauds phenomenonrenal disease as a complication in 50% of casesdeveloping after 12 to 24 monthsacute nephritic syndrome in 20-30% of renal diseaseoliguric acute renal failure in 50% of renal disease

acute nephritic syndrome and RPGNimmune-complex glomerulonephritiscryoglobulinemia glomerulopathyLab:depressed circulating levels of C3, C4 and CH50(80% of cases of renal involvement)transient ANA positivityhepatitis C (HCV) RNA has been isolatedTx:glucocorticoidswith or without cyclophosphamideplasmaphresisHCV infectioninterfeon prognosis:75% alive at 10yr

acute nephritic syndrome and RPGNanti-GBM diseaseGoodpasture syndromeusually as RPGN (crescentic glomerulonephritis)acute nephritic syndrome is rarepulmonary hemorrhage (50-70%) predates by wks to months2 peaks of patient population(1) young (5 to 40 yr) male (6M:1F)(2) sixth decade both sexes (less pulm hemorrhage)clinical Sx/Lab:hematurianephritic urinary sedimentsubnephrotic proteinuriarapidly progressive renal failure over weekswith or without pulmonary hemorrhage

acute nephritic syndrome and RPGNanti-GBM diseaseGoodpasture syndromeDx:circulating anti-GBM antibodiesrenal biopsy is the gold standardpathology:LM:crescentic glomerulonephritis- diffuse proliferative glomerulonephritis with focal necrotizing lessions and crescents in >50% of glomeruliIF:linear ribbon-like deposition of IgG along GBM

acute nephritic syndrome and RPGNanti-GBM diseaseGoodpasture syndromeTx:early and aggressive use of plasmapheresis, glucocorticoids, cyclophophamide and azothioprine is keyspeed of initiation therapy is a critical determinant of outcomeemergency plasmapheresisdaily or alternate days until no detectable anti-GBM(may take 1 to 2 wks)prognosis:requiring dialysis at presentation as a poor prognostic sign (no recovery of renal function)relapse not uncommon

acute nephritic syndrome and RPGNpauci-immune glomerulonephritisa spectrum of a single disease(ANCA-associated small vessel vasculitis)old (~57yr) whitenon-specific constitutional signs and symptomslethargyfevermalaisearthralgiaanorexiamyalgiawt. lossnon-specific lab abnormalitiesrapid sedimentation rateelevated C-reactive proteinnormochromic, normocytic anemianormal serum complement levelsleukocytosisthrombocytosis

acute nephritic syndrome and RPGNpauci-immune glomerulonephritisLM:focal, segmental, necrotizing glomerulonephritis with crescent formationIF, EM: paucity or absence of immunoglobulin, complement, and immune depositsTx:glucocorticoids and cyclophophamideazathioprine or mycophenolateplasmapheresisdialysisrenal transplant30% will relapse after treatment-induced remissiontrimethoprim-sulfamethoxazole reduces relapse rate

Complications:

Glomerular diseases:

Urine Microscopy :Cells Casts Crystals.Casts are formed within nephron.Casts Suggest Kidney pathology. Casts can be made up of Protein, lipid, cells or mixed. Crystals suggest high concentration or altered solubility.

Definisi: sindroma klinik dgn sejumlah kelainan renal & ekstra renal.Terdiri dari:Proteinuria (>3,0-3,5gr/hr)HipoalbuminemiaEdemaHiperlipidemiaHiperkoagulasi

Primer (idiopatik) 75-80%Sekunder : penyakit sistemik (DM,SLE), keganasan,toksin spesifik

Causes of Idiopathic Nephrotic Syndrome

Disease

Minimal Change Focal Sclerosis Membranous nephropathy Mrmbranoproliferative GN

Haas, et al. AJKD 30:621, 1997Korbet, Et al. AJKD 27:647,1996Children 70101510Adults

15353310

Minimal Change Disease

Focal Segmental

Membranous glomerulonephritis

Causes of the Nephrotic SyndromePRIMARY GLOMERULAR DISEASE- Minimal change disease- Focal segmental glomerulosclerosis - Membranous glomerulonephritis - Membranoproliferative glomerulonephritis

SECONDARY DISORDERS- Systemic diseases (DM,SLE)- Medications - Infections- Malignancy

Taken from the Clinical Nephrology on CD-ROM (Copyrights 1997, OUP) - Chapter 3.5, Figure 4: A diagram of the events pictured in the 'classical' or 'underfill' hypothesis of neph.--------------------------------Figure 4. A diagram of the events pictured in the classical or underfill hypothesis of nephrotic oedema. It is possible that this sequence is dominant in childhood patients developing oedema with sudden onset of severe proteinuria in a setting of minimal change disease, but this is an inadequate explanation for the continued presence of oedema in most nephrotic patients (see text for detailed discussion).

1. Minimal change disease (MCD)2. Focal segmental glomerulosklerosis (FSGS)3. Membranous glomerulopathy4. Membranoproliferatif glomerulosklerosis (MPGN)

Perlu biopsi untuk :Diagnosis defenitifPenatalaksanaanprognosis

Ditemukan pd 80% NS umur < 16 thn & 20 % pd dewasa Ukuran & arsitektur glomerulus relatif baik- Respon terhadap steroid : baik- Prognosis : baik

Minimal Change/Lipoid :

Loss of Foot processes

Terjadi pada 1/3 NS dewasa 50% glomerulus sklerosis & hyalinosis Respon steroid : 20-40 % kasus Remisi spontan : jarang Prognosis : buruk

Terjadi pada 30-40 % NS dewasaPatologi :penebalan diffuse basal membran glomerulus tanpa adanya inflamasi atau proliferasi sellulerTidak ada perbaikan dgn steroid

Patologi : Penebalan dan proliferasi memban basalis glomerulusAda 3 tipe:Tipe I : immune deposit pd sub endotel & mesangiumTipe II : AutoimmuneTipe III : kombinasi Tipe I & membranous glomerulopathyUntuk MPGN tidak ada terapi spesifik

Bila edema : rawat di RSDiet : protein 0,8gr/kgBB/hr garam dibatasiDiuretik : furosemid, spironolaktonPrednison : 1-1,5mg/kgBB/hr selama 4 mgg diteruskan 1 mg/kgBB/hr alternate day 4 mgg 90% remisi pd terapi 20-24 mgg

Sitostatika Cyclofosfamid 2-3 mg/kgBB/hr atauClorambusil 0,1-0,3mg/kgBB/hr selama 8-12 mggDipertimbangkan jk terjadi relaps > 3 kali/thn atauSindroma nefrotik yang steroid dependent

Taken from the Clinical Nephrology on CD-ROM (Copyrights 1997, OUP) - Chapter 3.5, Table 1: Inhibition of proteinuria in non-diabetic nephrotic patients by different agents.--------------------------------

* Angiotensin II plays a central role in renal disease. An activated reninangiotensinaldosterone system (RAAS) promotes both systemic and glomerular hypertension, which can induce haemodynamic injury to the vascular endothelium and glomerulus.1 Angiotensin II, the primary agent of the RAAS, is a mediator of a range of responses, most immediately blood pressure increase by angiotensin-induced renal vasoconstriction and the secretion of aldosterone which leads to increased sodium and water resorption. The role of angiotensin II in hypertension is complex and involves several feedback loops. When chronically elevated, angiotensin II leads to hypertension, which in turn leads to further activation of angiotensin II and establishes a feedback loop leading to further activation.2 In addition, direct profibrotic and proinflammatory actions of angiotensin II and aldosterone may also promote kidney damage.1 The AT1 receptor is responsible for most of the pathophysiological actions of angiotensin II.1

Brewster UC, Perazella MA. The renin-angiotensin-aldosterone system and the kidney: effects on kidney disease. Am J Med 2004;116:263272. Erhardt LR. Endothelial dysfunction and cardiovascular disease: the promise of blocking the renin-angiotensin system. Int J Clin Pract 2003;57:211218.

*TGF-Angiotensin II and Thrombospondin (TSP1) can both stimulate the production of transforming growth factor-b (TGF-) by tubuloepithelial cells and fibroblasts. TGF-, in turn, causes proliferation of fibroblasts and tubuloepithelial cells. TGF- ultimately increases extracellular matrix proteins, likely by several mechanisms. 1) TGF- stimulates production of several growth factors including basis fibroblast growth factor (bFGF) and platelet derived growth factor (PDGF) that stimulate the formation of extracellular matrix (ECM) proteins. 2) TGF- also has a direct effect on ECM protein expression. 3) TGF- stimulates the formation of the receptors to which ECM proteins adhere. 4) TGF- increases the expression of tissue inhibitors of metalloproteases (TIMP) that, in turn, prevents proteases from degrading ECM proteins. 5) TGF- stimulates the endothelial cell production of endothelin-1 (ET-1). The effects of ET-1 are illustrated on a separate slide.

References: Klahr S and Morrissey JJ. The role of vasoactive compounds, growth factors and cytokines in the progression of renal disease. Kidney Int 2000;57[Suppl75]:S7-14.

Inoki K, Haneda M, Ishida T, Mori H, Maeda S, Koya D, Sugimoto T, and Kikkawa R. Role of mitogen-activated protein kinases as downstream effectors of transforming growth factor-beta in mesangial cells. Kidney Int 2000;58[Suppl 77]:76-80.

*TGF-Angiotensin II and Thrombospondin (TSP1) can both stimulate the production of transforming growth factor-b (TGF-) by tubuloepithelial cells and fibroblasts. TGF-, in turn, causes proliferation of fibroblasts and tubuloepithelial cells. TGF- ultimately increases extracellular matrix proteins, likely by several mechanisms. 1) TGF- stimulates production of several growth factors including basis fibroblast growth factor (bFGF) and platelet derived growth factor (PDGF) that stimulate the formation of extracellular matrix (ECM) proteins. 2) TGF- also has a direct effect on ECM protein expression. 3) TGF- stimulates the formation of the receptors to which ECM proteins adhere. 4) TGF- increases the expression of tissue inhibitors of metalloproteases (TIMP) that, in turn, prevents proteases from degrading ECM proteins. 5) TGF- stimulates the endothelial cell production of endothelin-1 (ET-1). The effects of ET-1 are illustrated on a separate slide.

References: Klahr S and Morrissey JJ. The role of vasoactive compounds, growth factors and cytokines in the progression of renal disease. Kidney Int 2000;57[Suppl75]:S7-14. Inoki K, Haneda M, Ishida T, Mori H, Maeda S, Koya D, Sugimoto T, and Kikkawa R. Role of mitogen-activated protein kinases as downstream effectors of transforming growth factor-beta in mesangial cells. Kidney Int 2000;58[Suppl 77]:76-80.

*TGF-Angiotensin II and Thrombospondin (TSP1) can both stimulate the production of transforming growth factor-b (TGF-) by tubuloepithelial cells and fibroblasts. TGF-, in turn, causes proliferation of fibroblasts and tubuloepithelial cells. TGF- ultimately increases extracellular matrix proteins, likely by several mechanisms. 1) TGF- stimulates production of several growth factors including basis fibroblast growth factor (bFGF) and platelet derived growth factor (PDGF) that stimulate the formation of extracellular matrix (ECM) proteins. 2) TGF- also has a direct effect on ECM protein expression. 3) TGF- stimulates the formation of the receptors to which ECM proteins adhere. 4) TGF- increases the expression of tissue inhibitors of metalloproteases (TIMP) that, in turn, prevents proteases from degrading ECM proteins. 5) TGF- stimulates the endothelial cell production of endothelin-1 (ET-1). The effects of ET-1 are illustrated on a separate slide.

References: Klahr S and Morrissey JJ. The role of vasoactive compounds, growth factors and cytokines in the progression of renal disease. Kidney Int 2000;57[Suppl75]:S7-14. Inoki K, Haneda M, Ishida T, Mori H, Maeda S, Koya D, Sugimoto T, and Kikkawa R. Role of mitogen-activated protein kinases as downstream effectors of transforming growth factor-beta in mesangial cells. Kidney Int 2000;58[Suppl 77]:76-80.