La Nefrologia tra passato,presente e

futuro

Antonio Santoro

Nephrology, Dialysis and Hypertension Unit

Sant’Orsola-Malpighi Hospital

Bologna - ITALY

University Hospital

• The ultimate goal of a nephrologist is to maintain renal

function and to treat kidney diseases,manage

associated metabolic changes and prolong time till

dialysis.

• Nephrologist perform hemodialysis,PD, and other

extracorporeal depuration techniques.

• Nephrologists manage Acute Renal Failure and provide

lifesaving Continuous and intermittent Renal

Replacement Therapy (CRRT).

• Nephrologists are specialists in electrolyte, fluid

balance, acid/base, anemia assoc renal disease,

metabolic bone disease, hypertension management

and renal trasplantation.

The nephrologist

J. Stewart Cameron 1982

Machuca, E. et al. Hum. Mol. Genet. 2009 18:R185-194R

Glomerular filtration barrier structure

Machuca, E. et al. Hum. Mol. Genet. 2009 18:R185-194R

Molecular overview of the slit-diaphragm and podocyte cell-matrix interactions

Various molecules having diverse functions at the glomerular filtration

barrier may be affected by mutations in genetic forms of NS.

However, recent findings have indicated that different pathogenetic

mechanisms have converging lines in that the rearrangement of the

podocyte’s actin cytoskeleton has been identified as a common

denominator of a variety of inherited glomerular disorders.

It has also become evident that the molecular sieve formed by the

glomerular filtration barrier is not a static structure. In fact, the podocyte

layer has emerged as a particularly dynamic network of

interacting cells that contain an actin-based contractile apparatus.

It seems likely that the podocytes’ ability to continuously sense

and adapt to environmental changes is critical for preserving

glomerular permselectivity. Notably, there is emerging evidence that also

podocyte injury of non-genetic cause may involve the same

pathogenetic pathways, as it is associated with reorganization of the slit

diaphragm and of the foot process structur

Despite common wisdom, the role of essential hypertension in the etiopathogenesis of ESRD has

been controversial. Two recently published studies demonstrated a strong association of genetic

variants in the gene that encodes the molecular motor protein nonmuscle myosin 2a (MYH9) with

ESRD in African American patients without diabetes. These new data demonstrate that much of

the excess risk of ESRD in African American individuals is attributable to an MYH9 risk

haplotype and suggest that hypertension may cause progressive kidney disease only in

genetically susceptible individuals or be the result of a primary renal disease.

Genetic dissection of T2DM-associated ESRD reveals that MYH9 underlies a portion of this

clinically diagnosed disorder in African Americans. It is likely that a subset of African

Americans with T2DM and coincident nephropathy have primary MYH9-related kidney

disease (e.g. FSGS or global glomerulosclerosis)

… altri contributi della genetica ….

Diagram of the PKD1 protein, polycystin-1 (left) and the PKD2 protein,

polycystin-2, and their interaction through coiled-coil domains in the C-

terminal tails (according to the revised molecular model)

Torres V et al KI 2009

Diagram depicting putative pathways up- or downregulated in polycystic

kidney disease and rationale for treatment with V2 receptor antagonists,

somatostatin, triptolide; tyrosine kinase, src, MEK, TNF , mTOR, or CDK

inhibitors; metformin, and CFTR or KCa3.1 inhibitors

Clin JASN 2008

NDT 2009

2008

CURRENTLY ACTIVE TRIALs for ADPKD

Torres, V. E. Clin J Am Soc Nephrol 2008;3:1212-1218

Kidney sections from PCK rats, Pkd2-/WS25 mice, and pcy mice untreated (top) or treated

(bottom) with OPC-31260

Diagnostic tools

1960 1970 1980 1990 2000

Chest X Ray

ECG

Nuclear Medicine

Echo

Angiography

CT

Doppler Echo

TEE

MRI

PET

MDCT

Chest x ray

SPECT

CT

MRI

PET

MDCT

Echography

Coronary angiography

TEE

IVUS

Diagnostic tools

EBCT

$

Spectroscopy

Multidetector CT

evolution or revolution? (Rubin G, Radiology 2000)

http://i378.photobucket.com/albums/oo221/miloncho/rxuro2.jpg

Cellule staminali

Ronconi, E. et al. J Am Soc Nephrol 2009;20:322-332

Schematic representation of the hierarchical distribution of CD133+CD24+PDX- and CD133+CD24+PDX+ cells within human

glomeruli

Ronconi, E. et al. J Am Soc Nephrol 2009;20:322-332

Heterogeneous expression of renal progenitors and podocytes markers by cells of Bowman's capsule in adult human kidney

Urinary proteomics

Test Sensitivity Method Advantages Disadvantages

Urine Dip >250 mg/l Binding of urine protein to

tetrabromophenol blue

Quick and cheap Lower limit of sensitivity, some

proteins do not bind e.g.

Bence–Jones protein

24 h urine

collection

30 mg/24 h Biuret method Current gold-standard Inaccurate due to incomplete

collection, inconvenient and

time consuming

Protein/cre

atinine

ratio

0.02

mg/µmol

Corrects for variation in urinary

concentration due to hydration

Simple, cheap and quick,

replacing 24 h urine

collection

Only measures total protein

Immunoglo

bulin light

chains

0.04 g/l Urine protein electrophoresis

and immunofixation or

immunoassay-based free light

chain assay

Able to detect at low levels Expensive and disease

specific

Current methods for assessing proteinuria

Bramham, K. et al. QJM 2009 102:523-538

Schema of translation of urinary proteomics into clinical practice

Recent developments in proteomic techniques promise exciting insight

into normal renal physiology and renal disease processes, allowing

precise definition of the disease proteome. In turn this will enable

diagnosis, prognosis and prediction of response to treatment of

individuals. …………………………………............…

Expanding knowledge in recent years and the rapidly advancing

field of proteomics will facilitate studies of renal physiology and

pathophysiology and ultimately should make redundant the

traditional renal biopsy.

QJM 2009 102(8):523-538

Characterization of Diabetic Nephropathy by Urinary Proteomic

Analysis: Identification of a Processed Ubiquitin Form as a Differentially

Excreted Protein in Diabetic Nephropathy Patients Hassan Dihazi et al. Clinical Chemistry 2007; 53: 1636-1645

Ubiquitin degradation assay

In summary, in this pilot study we identified 3 different proteins that were differentially excreted in the urine of

diabetic nephropathy patients compared with the other groups. A processed form of ubiquitin with m/z 6188

was missed in the urine of diabetic nephropathy patients.

This ubiquitin form could be used as a prognosis marker for DM-NP. Quantification of this protein

during the progressive disease course in the DM-NP patients will give interesting information on the

development of the diseases and serve as a good marker for prognosis.

1854: The Scientific Basis for Dialysis

Script of Graham´s Bakerian Lecture, 1854

Thomas Graham

1805 - 1869 “... might be

applied to

medicine ...”

1913: A First “Artificial Kidney”

Abel JJ, Rowntree LG, Turner BB: On the removal of

diffusible substances from the circulating blood by

means of dialysis. Trans Ass Am Physicians 28:51, 1913

John Jacob Abel

1857 - 1939

"... by which the blood of a living animal may be submitted to

dialysis outside the body, and again returned to the natural

circulation without exposure to air, infection by micro-organisms or

any alteration which would necessarily prejudicial to life."

burette with hirudin solution

1913 - 1937: Anticoagulation as a Major Problem

Before 1916 hirudin extracted from the salvia

of leeches was the only practical

anticoagulant. Insufficient purification caused

major side effects and allergic reactions.

Hirudin was first identified in 1884.

In 1916 McLean isolated heparin as a

naturally occurring mammalian anticoagulant.

Haas (see next slide) used heparin in animal

experiments.

Use of heparin in human medicine became

a standard after appropriate purification

technology was available in 1937 (Murray et.

al).

Recombinant hirudin currently experiences a

come-back.

1945: The First Surviving Patient (1)

Willem Kolff Kolff´s rotating drum

dialyzer (1943, Kampen,

Netherlands)

Timeline: Major Achievements in Hemodialysis

1850 1860 1870 1880 1890 1900 1910 1920 1930 1940 1950 1960 1970 1980 1990 2000

Diffusive membrane

transport

First anticoagulant

(hirudin)

First animal

dialysis

First human

dialysis

First patient

surviving

due to

dialysis

First chronic

HD program

First high flux

dialyzer & related

machine

First single pass

machine for all

types of dialyzers

EPO

Polysulfone®

Natural occurring

mammalian anticoagulant

(heparin) for medical

application

Possibili usi del Personal Computer in una divisione di nefrologia e dialisi.

Santoro A. et. Al.

Attualità nefrologiche e dialitiche San Carlo 1984

– Patient archiving for administrative

purposes

– Computerised monitoring of

dialysis parameters

– Computerised clinical records

– Modelling and simulation

– Computerised control of dialysis

equipment

Automazione in dialisi

L’automazione come

informatizzazione di dati clinici e dati

macchina

L’informatica come controllo di

processi

L’ informatica nella gestione della

terapia dialitica : I biofeedbacks

Networking and connectivity

PC03 PC02 H

U

B

Local Network

Physician

Hospital Dialysis Centre Router 1

Router 2

Limited Care Centre

SERVER

PC01

Possibilità Attuali di

Monitoraggio della

Dialisi

• WHY ?

• WHEN

?

• HOW ? …. and what kind of results ?

Il monitoraggio in tempo reale

permette :

• di mantenere sotto continuo controllo

parametri fisiologici,biochimici ed

emodinamici

• di prevenire situazioni critiche

• di modificare in anello aperto o con

feedback automatici attuatori dialitici

PARAMETRI SUSCETTIBILI DI

MONITOROGGIO ON-LINE

Efficienza dialitica (Kt/V)

Volume Ematico

Stabilità pressoria

Ricircolo dell’accesso

Bilancio termico

Controllo automatico dei parametri fisiologici ... Un “pilota automatico” per l’emodialisi

HD

system

Dialysate

sensors

Blood

sensors

Patient

sensors

Modules

Monitor

Hydraulics

─ Beta2 amyloidosis

─ LVH

─ Neuropathy

─ Anemia

─ Malnutrition

─ Accelerated artheriosclerosis

─ Cardiovascular calcifications

─ Cardiovascular instability

─ Immunological depression

─ Infections

─ High mortality

Conventional HD

DIALYSIS MEMBRANE EVOLUTION

• Low-flux membranes

• High-flux membranes (pore size & water permeability)

• removal of MM

• removal of low MW proteins

• Superflux or Protein-leaking membranes • clearance for low MW prteins & small prrotein-bound

solutes (Homocystein, AGEP…)

High Cut-Off membrane

Cut-off ≈ 60.000 Dalton Increase in:

pore size

homogeneous pore distribution

Increase in membrane permeability

Transfer of protein-bound solutes

SUPERFLUX DIALYZERS

MAXIMISATITON OF THE CONVECTIVE

TRANSPORT BY INCREASING THE PRESSURE

DROP ALONG THE FIBERS

INCREASE IN PORE SIZE

INCREASE IN THE HOMOGENEOUS DISTRIBUTION

OF PORES

INCREASE THE MEMBRANE PERMEABILITY

TRANSFER OF LARGE TOXINS (PROTEIN-BOUND)

Dialyzer characteristics

Low-flux

polysulphone

(F60)

Low-flux triacetate

(Sureflux 150L)

High-flux triacetate

(FB-150U)

Super-flux

triacetate

(Sureflux 150FH)

Super-flux

polysulphone

(F500S)

Effective surface

area m2 1.3 1.5 1.5 1.5 1.2

Inner diameter, m 200 200 200 185 155

Membrane

thickness, m 40 15 15 15 35

Pore size, A 50 70 78

Ratio of open pores,

% 63 70 84

Sieving coefficient

2MG

0.65 0.36 0.88 1 0.9

UF coeff,

ml/mmHg,h 40 19 30 67 300 (H2O)

Van Tellingen A et al NDT 2004

25

20

15

10

5

0

-5

-10

-15

-20

-25

Plasma leptin

(ng/ml)

Week 1 week 12 week 1 week 12 week 1 week 12 week 1 week 12

F 6HPS TRICEA 15 G F 60S F 500S

Superflux dialyzers on leptin levels

Superflux

F500S

MW 160000 D

INFLAMMATORY MEDIATORS IN CONVENTIONAL CVVH

Mediator Molecular Weight (daltons)

Sieving Coefficient Clearance (ml/min)

Bradykinin 1,100

Endothelin 2,500 0.19

C3a / C5a 11,000 0.11-0.77

Factor D 24,000

LPS 67,000

LPS fragments < 1000 – 20,000

TNF- 50,000 0-0.2 3.6

sTNFr 30,000 – 50,000 <0.1

IL-1 17,500 0.07-0.42

IL-1ra 24,000 0.28-0.45 13.2

IL-6 22,000 1.2

IL-8 8,000 0-0.48 4.4

IL-10 18,000 0

INF- 20,000

Morgera S et al. Crit Care 2006

HIGH-CUTOFF HEMOFILTRATION IN

SEPSIS

0

200

400

600

800

1000

0

200

400

600

800

1000

0

1000

2000

3000

0

1000

2000

3000

p=0.0465

High cut off hemofilter (P2SH) Conventional hemofilter (PF 11S)

p=0.5506

p=0.0014 p=0.7789

Inte

rleu

kin

-6 [

pg/m

l]

Inte

rleu

kin

-1ra

[p

g/m

l]

Inte

rleu

kin

-1ra

[p

g/m

l]

Inte

rleu

kin

-6 [

pg/m

l]

High cut off hemofilter (P2SH)

Conventional hemofilter(PF 11S)

0 0

0 0 48

48 48

48

Time (hours)

Time (hours) Time (hours)

Time (hours)

SUPERFLUX FILTERS IN RHABDOMYOLYSIS

Naka T. Critical Care 2005,

Filter type UFR (L/h) Myoglobin Myoglobin

UF conc (g/L) removal (g/day)

Conventional 2 23.003 1.1

Superflux 2 >100.000 >4.8

Superflux 3 60.912 4.4

Superflux 4 53.527 5.

Meyer T W et al. Kidney Int 2004

Effect of protein binding on dialyzer solute clearance

REMOVAL OF FREE CIRCULATING LIGHT CHAINS (LC) BY

A HIGH CUT-OFF MEMBRANE: DIFFERENT DIALYSIS STRATEGIES

Sonia Pasquali , Elena Mancini, Antonio Santoro

and Collaborative Study Group on TheraliteTM HCO1100 in Myeloma.

Nephrology, Dialysis and Hypertension, Policlinico S.Orsola-Malpighi,

Bologna, Italy.

Eleven MM patients with high serum FLC levels (6 lambda/5 kappa; FLC=8,9±5,5 g/L),

were enrolled in an acute study where every patient was his/her own control.

HD HDF

Duration (h) 8 8

Filter Theralite HCO1100TM

QB (ml/min) 200 200

QD (ml/min) 500 500

QINF (ml/min) 0 33

Vascular Access Artero-venous fistula or dual lumen catheter (13F)

Heparin strategy Initial bolus, then continuous inrfusion, flow rate adjusted to

achieve the desired activated coagulation time (ACT, HemocronR)

Assessment of serum

and dialysate FLC

(FreeliteTM assay)

Basal, after 30, 60, 120, 240, 480 mins. and after 60 mins after end

of treatment.

Ultrafiltration Diffusion Convection Adsorbtion Plasmafiltration

BLOOD PURIFICATION PROCESS & THERAPIES

SCUF CVVHD CVVH PEX HP

CVVHDF

CPFA

Selective Plasmapheresis

Cardiology

•Congestive heart failure

•Pulmonary Hypertension

→Slow Continuous Ultrafiltration

Hematology

•Myeloma

•Tumor lysis syndrome

→Plasma-exchange

→Superflux dialysis

→Plasmapheresis Hepatology

•Acute on Chronic Liver Failure

•Acute liver failure

•Hyperbilirubinemic states

→Plasma-exchange

→Plasmapheresis (selective)

→Hemoperfusion

Toxicology

•Intoxications

•Poisoning

→Dialysis (Lithium, Ethilen Glycol, Bromure. Theofillina…)

→Hemo/Plasmaperfusion (Barbiturates, Salicilates, Glutetimide, Paraquat)

The “Present-Future”

Pathways to Renal Replacement Therapy

Undiagnosed

Community

CKD

Known

Community

CKD

Kidney

Care

CKD or AKI in

Secondary Care

Centre/Satellite

HD/HDF/HF

Home HD

PD

Conservative

Care

Late

Referral

Timely

Referral

AKI

Trasplant

Conv. HD

Nocturnal

HD

Daily HD

6 times/week

HD (4 hrs.)

6 times/week

HD (8 hours)

3 times/wek

8 hours

WAK

The universe of innovations in dialysis

Different HD

schedules:

Frequency, Duration

Dialysis location:

In-center? Satellites ?

At home? WAK ?

Diffusion? Convection?

How much convection?

Absorption

Molecola di eparina

Idrogel (acrilonitrile metallil sulfonato di sodio)

Eparina (3000 IU/m2)

Funzionalizzazione: Polietileneimmina ad alto peso molecolare

Membrana

• Attività anticoagulante mantenuta durante l’intera

sessione di dialisi (reversibilità dell’affinità eparina-TAT): – L’eparina esercita la sua attività anticoagulante legandosi

all’ATIII (anti-trombina III) – La configurazione di ATIII viene modificata dall’eparina – Si forma il complesso TAT che viene rilasciato nel circolo ematico – L’eparina rimane fissata alla membrana (alta affinità)

Bioattività dell’eparina graffata:

Membrana

CELL THERAPY, THE BIOARTIFICIAL

KIDNEY (BAK)

Tulim et al. JASN 2008

• Immunoregulatory role of tubule cells:

– Glutathione peroxidase synthesis

– Glutathione reclamation

– Activation of Vit D3

• Immunological actions of proxymal tubular cells:

– Antigen-presenting cells

– Synthesis and process of inflammatory cytokines

Propensity of patients with ARF to develop SIRS and sepsis

Renal tubule cell therapy: potential mechanisms of

action in ARF

0

1

2

3

4

5

6

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

BAK in animals: effect on Gram- negative induced

septic shock

12 nephrectomized dogs; 48 h later, 30x1010E.Coli/Kg; CVVH + Sham RAD (n=6) or

Cell-RAD (n=6); 24 h treatment

Survival Time:

RAD animals: 13.0+0.7 h

Sham controls: 9.7+0.5 h

P<0.02

Time (hours)

Cardiac Output (L/min)

Cell RAD

Sham RAD

0

20

40

60

80

100

120

140

160

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Time (hours)

Systolic Blood Pressure

(mmHg)

Cell RAD

Sham RAD

Modified from:

Fissel WH et al, JASN 2003

Tulim J et al. JASN 2008

BAK in humans: efficacy and safety, phase II

clinical trial

Multicenter RCT, 58 pts with ARF requiring CRRT

Primary outcome: 28 days all cause-mortality

Additional outcomes:

Mortality at 90 & 180 days

Time to recovery of renal funciton

Time to discharge

Safety

40 pts CRRT+RAD

18 pts CRRT alone

Completed the study: 25/58

•RAD group: 21/40 (53%)

•CRRT group: 4/18 (22%)

Time point

RAD (n=40; n [%]) CRRT Alone (n=18; n [%])

P* Died Alive Unknowna Died Alive Unknown

28 d 13 (33.3) 26 (66.7) 1 11 (61.1) 7 (38.9) 0 0.0821

90d 15 (38.5) 24 (61.5) 1 12 (66.7) 6 (33.3) 0 0.0855

180 d 18 (50.0) 18 (50.0) 4 13 (76.5) 4 (23.5) 1 0.0817

a * P values from exact Pearson X2 test; analysis excludes patients with unknown status.

Tulim J et al. JASN 2008

BAK in humans: survival

HR for death, adjusted for disease cause:

RAD group vs CRRT alone:

0.481 (CI 0.23-0.99)

La Dialisi Peritoneale è una DIALISI

DOMICILIARE

Può essere eseguita con modalita’

manuale (CAPD)

automatizzata (APD)

E’ rivolta a :

Pazienti in grado gestire autonomamente

il trattamento

Pazienti affiancati da un partner

efficiente LA DIALISI PERITONEALE PRESUPPONE LA

PRESENZA DI GRANDE AUTONOMIA

GESTIONALE

GRANDE CONTROLLO

CENTRALE

1. L’ icodestrina è un polimero del glucosio che viene

idrolizzato a maltosio a livello intraperitoneale.

2. Capacità di ultrafiltrazione elevata e prolungata nel

tempo, grazie ad una ridotta back-diffusion dalla cavità

peritoneale alla circolazione sanguigna

3. Basso contenuto di glucosio e conseguentemente ridotta

produzione di prodotti di degradazione del glucosio

4. Monoscambio quotidiano, con notevole riduzione dei

rischi legati alla manovra per se

L’ICODESTRINA

1200

900

600

300

0

UF netta (ml)

1,5% 4,25% 7,5% 7,5%

Paz. con alta UF

Paz. con bassa UF

Pannekeet MM et al, KI 1996;50:979-986

Glucosio

Icodestrina

Years Years

Years

2 4 6 8 10 0

20%

0%

40%

60%

80%

100%

1981 - 1985

Graft Survival by Era

Kidney trasplantation

73

Trapianto di Rene e Rigetto Acuto

Zand M, Seminars in Dialysis, 2005

0

40

80

100

%

‘60 ‘65 ‘70 ‘75 ‘80 ‘85 ‘90 ‘95 ‘00 ‘05

Years

20

60

Rejection < 12 mo

74

Immunosoppressione Clinica e Trapianto

Steroidi

Azatioprina

Abs Policlonali

Ciclosporina

OKT3

Tacrolimus

Micofenolato

Rapamicina

Everolimus

Ab Monoclonali

Thymoglobulne

1955 1960 1970 1980 1990 1995 2000

Myfortic

Advagraf

CP 690-550

FTY 720

Alemtuzumab

Rituximab

Belatacept

AEB

……..

2004 2008

Antigen Presenting Cell

MHC II

T Linfocita

mTOR

CD 40 CD 80

86

CD 3

G1

M

G2

S

GO AP1 NFKB

mRNA

Calcineurina

CD 28 CD 154

IL- 2 IL- 15

CD 25

NFAT

CD52 JAK3

1 3

2

S-1-P

receptor

CD 122

Sintesi

nucleotidi

PKC Steroidi

LFA-3

CD 2

Azatioprina

Ciclosporina

Tacrolimus

AntiCD3 mAb

(OKT3)

Basiliximab

Daclizumab

Alemtuzumab

(Campath)

FTY720

Micofenolato mofetile

Acido micofenolico

Sirolimus

Everolimus

Thymoglobuline

Trapianto d’Organo

Attivazione Linfocitaria

Nanoscienza

Biomimetica

Nanotecnologia

Nanomedicina

Nanostrutture

La nanotecnologia è un ramo della scienza applicata e

della tecnologia che si occupa del controllo della

materia su scala dimensionale inferiore al micrometro

(in genere tra 1 e 100 nanometri) e della progettazione

e realizzazione di dispositivi in tale scala.

Il termine "nanotecnologia" indica genericamente la

manipolazione della materia a livello atomico e

molecolare.

Rappresentazione al

computer di un "ingranaggio"

di dimensione nanometrica

J Biolog Chem, 2005

Shen LJ, Int J Nanom, 2007

Panchapakesan U, Int J Nanom, 2007

Pickup J, Diab Metab researc Rev, 2008

Realistic Perspectives

Cli

nic

al E

ffecti

ven

ess

Acceptable range

Minumum

standard

Cost

Achievable based on local

resource and priorities

Best practice: optimum clinical

and cost effectiveness

Gold standard: maximal clinical

but reduced cost effectiveness