Nephrogenesis

NephrogenesisNephrogenesis

Maria E. Ferris, MD, MPH

September 2001

OrganogenesisOrganogenesis

• Most parenchymal epithelial organs follow a simple scheme of embryonic organogenesis. An epithelial sheet or tube that is derived from one of the primordia, enters a process of sequential

branching to generate a treelike structure.

• In the kidney, the epithelial tube is to become the arborizing nephric duct-derived collecting duct system.


• A series of morphogenetic and differentiation events that starts with inductive interactions between 2 different primordial tissues and leads, in one of two mainstream processes: – formation of mesenchymal condensations

– formation of aggregates

Nephrogenic StagesNephrogenic Stages

• The kidney is derived from two different early embryonic tissue primordia: – The nephric duct the mesonephric duct

and continues through the Wolffian duct stage to the ureteric bud.

– The nephrogenic cord, after inductive signaling with the pronephric duct-derived cells the nephroi of mesonephros and

metanephros

Successive Bilateral Ontogenic StagesSuccessive Bilateral Ontogenic Stages

• Pronephros

• Mesonephros

• Metanephros

PronephrosPronephros

• Occurs at the 3rd gestational week

• About 7 tubules coalesce to form the pronephric duct

MesonephrosMesonephros

• 4th Gestational week

• 40 tubules that coalesce

• Production of a urine-like substance

• A few mesonephric tubules persist in males to form the epididymis, duct deferens and the ejaculatory duct

MetanephrosMetanephros

• Weeks 5-12 of gestation

• Lasts until 38 weeks of gestation

• Develops from 2 sources – The ureteric bud Ureter– The blastema Induces dichotomous

branching of the ureteric bud to form the collecting ducts, calyces and renal pelvis


• Occurs from 2 distinct embryological origins– the ureter-derived collecting duct

– the mesenchymal blastema which will form the nephrons (glomerulus to the junction of connecting tubule to collecting tubules)

• The ureter-derived collecting duct is induced to branch, while the mesenchymal blastema is induced to enter the critical process of mesenchyme-to-epithelium conversion or transition (MET).

Embryonic precursors of metanephros. Rudimentary pronephros, transiently functioning mesonephros, and permanent metanephros are sequentially induced and formed, thus recapitulating phylogeny of excretory system. This embryonic continuity also pertains to some transcription factors and signal molecules. [Modified from Horster M. Physiological Reviews 1999; 79:1157-91]

Histology of early metanephrogenic organization. Section through human kidney (~20.5 mm embryo) showing structures derived from Wolffian duct and metanephrogenic blastema in outermost zone of cortex. Peripheral branch of ureteric tree extends distally into an ampulla. Metanephric blastema has been induced to enter nephrogenic pathway and nephron anlage has completed mesenchyme-to-epithelium transition .

Mesenchyme-to-Epithelium Transition (MET)Mesenchyme-to-Epithelium Transition (MET)

• These events are:– epithelial cell polarization and

– differentiation into the highly specialized epithelial cell populations of the nephron

Signaling MoleculesSignaling Molecules

• Each step along the metanephrogenic pathway is initiated and organized by signaling molecules that are locally

secreted polypeptides, encoded by different gene families and regulated by transcription factors

Functional complexes in transition of mesenchymal to epithelial cells (MET). Nephrogenic mesenchymal cells (top) are induced to enter MET whereby several systems with signaling functions are activated. CAM-mediated signals and ECM-mediated signals interact with secreted growth factors to express epithelial phenotype (bottom).


• Proceeds from the medulla to the outer cortex, directed by the ductal branching of the ureteric bud-derived collecting tubule

Microculture of metanephrogenic unit. To study MET, a nephrogenic unit as defined by a single ureteric bud with induced adherent mesenchyme is transferred in collagens. Schematic view illustrates in vitro MET and early nephrogenesis.These processes are documented by electron microscopy and by molecular analysis. Noninduced mesenchymal cells enter apoptosis, and ureteric bud cells proliferate and migrate to form a monolayer

Nephrogenesis ModelNephrogenesis Model

• Set up in the 1940’s, at the NIH demonstrated in an organ system in vitro that – Kidney rudiments when removed at embryonic day

11 (E11mouse) follow an almost normal developmental program in culture,

– The isolated ureteric bud cannot develop without contact to the metanephric mesenchyme, and

– The isolated metanephrogenic mesenchyme can be induced to go through the MET by a number of tissues, (embryonic spinal cord & the ureteric bud)

Regulation of Developmental PathwaysRegulation of Developmental Pathways

• The nephrogenic mesenchyma and the ureteric bud are regulated by – Transcription factors and proto-oncogenes, – Polypeptide growth factors acting as

signaling molecules, and their receptors.

Modulation of Developmental PathwaysModulation of Developmental Pathways

• Modulated by cell adhesion molecule (CAM) complexes and their associations with the cytoskeleton, by extracellular matrix (ECM) glycoproteins and ECM receptor molecules such as the integrin

family, and by ECM degrading proteases.

Growth RegulationGrowth Regulation

• Proto-oncogenes that encode for receptor tyrosine kinases are involved in mesenchymal (nephrogenic)-epithelial (ductopenic) interactions

• Proto-oncogene encoded tyrosine or serine/threonine kinase, is the ureteric receptor for signaling molecules secreted by the metanephrogenic mesenchyme

• Proto-oncogenes regulate growth and have the potential to gain tumorigenesis after gene mutations (Wilms tumor).

Genetic SignalsGenetic Signals

Temporospatial expression of signaling systems in nephrogenic and ureteric bud morphogenic pathways. Receptor tyrosine kinases (Ret, Met, Ros) are encoded by proto-oncogenes. Growth factor signaling molecules are expressed and secreted as indicated. Relative abundance of expression is specified by bold or normal type. Induced pre-condensing mesenchyme is shown on top; other stages of metanephric nephrogenesis correspond to those depicted highly schematically in Fig. 3, A, C, and E. PDGF, platelet-derived growth factor.

Overview of principal events in early nephrogenesis. Ureteric bud, an offspring of Wolffian duct, invades mesenchymal blastema (left) and initiates reciprocal signaling (middle) between epithelial (ductal) and mesenchymal (metanephrogenic) cell types. Receptor tyrosine kinases are expressed almost exclusively in ureteric bud cell, whereas ligands are secreted by adjacent mesenchymal cells. Ligand for c-ros encoded receptor is not yet known. [Horster, M. Physiological Reviews. 1999; 79: 1157-91]

Patterns of expression and repression of critical developmental genes. Genes that code for transcription factors and for signaling molecules interact positively (expression) or negatively (repression) or behave autoregulatory. Stages of metanephric morphogenesis require profound changes in gene expression, for cell condensation and adhesion, MET, epithelial cell apicobasal polarization, nephron segmental pattern formation, and acquisition of membrane transport molecules. Regulation of most expression events and downstream gene targets remains elusive.

Gene Expression Pattern Renal Murine Phenotype

WT-1 Uninduced mesenchymeNo ureteric bud outgrowth, no induction ofmesenchyme

Wnt-4 Mesenchymal condensateArrest of nephrogenesis at condensation stage,but initial branching of ureteric bud

Pax-2Induced mesenchyme, Wolffianduct, ureteric bud

Wolffian duct growth blocked and metanephrosdeleted

c-ret Ureteric bud tip cells Branching morphogenesis blocked

lim-1 Mesenchymal condensatesAbsence of metanephros and precursors and ofWolffian duct

ld Mesenchyme, ureteric budUreteric bud outgrowth blocked, no induction ofmesenchyme, renal agenesis

Mouse mutants, occurring naturally or engineered genetically, have contributed much tounderstanding the roles of developmental genes. Wild-type expression pattern andphenotype after targeted mutation of a few developmental genes are listed.

Gene Mutations Influencing NephrogenesisGene Mutations Influencing Nephrogenesis

GROWTH FACTORS AND EXTRACELLULAR MATRIX

A. Growth Factors Are Signaling Molecules in Induction and Differentiation

B. Growth Factor Families Are Expressed in Temporospatial Patterns

C. Extracellular Matrix Proteins (ECM) and Cells Interact in Epithelial Morphogenesis

A. Growth Factors are Signaling A. Growth Factors are Signaling MoleculesMolecules• In addition to their mitotic (growth) action, not only

mediate motogenic (migration) & morphogenic inductive

signals, but also those for cell differentiation (polarization), proliferation, and apoptosis.

• The roles of the growth factor families and their receptors, (each a multigene family) is very complex.

• The best-characterized growth factors pertinent to renal organogenesis are IGF-I and IGF-II, HGF, TGF- and FGF

B. Growth Factor Families Are Expressed in Temporospatial Patterns

• 1. IGF-I and IGF-II

• 2. HGF (hepatocyte growth factor)/SF

• 3. TGF- (Transforming growth factor)

• 4. TGF- /EGF

• 5. PDGF (Platelet-derived Growth Factor A & B)

• 6. Bmp 7 (bone morphogenic protein)

• 7. NGF( Nerve growth factors) & neurotrophin-3

C. ECM and Cells Interact in Epithelial Morphogenesis

• Most ECM molecules contain multiple binding domains that are recognized to interact with integrins, their glycoproteins have signal transducing receptors & they mediate events such as cell adhesion in embryonic cells

– ECM molecules have morphoregulatory functions

– Laminins transfer ECM signals to the cell

– Integrin expression repertoire changes with nephrogenesis

GENES THAT CONTROL NEHPROGENESIS

• A. Transcriptional Regulation – Wilms Tumor Suppressor 1

– Paired-box genes (Pax-2)

– BF-2 and the stromal cell lineage

– Homeobox genes (Hoxd-3)

– Hepatocyte nuclear factors

– The myc gene family

GENES THAT CONTROL NEHPROGENESIS

• B. Signaling by Receptor Tyrosine Kinases– C-met

– C-ret

– C-ros

GENETIC ERRORS IN NEPHROGENESIS

• Polycystic Kidney Disease

– Dysruption of C-myc and bcl2 on ARPKD

– Polycystin may participate in the epithelial polarization process following MET.

• Wilms Tumor

– Suppressing activity of WT-1 might be cause erroneous blastemal growth

• Renal Cell Carcinoma

– Reexpression of Pax-2 in mature epithelium may be a predisposition for oncogenesis

Newborn GFRNewborn GFR

• In utero is the cardiac output to the kidney is low (2-4%) due to placental function

• Increase in renal mass between 35-40 wks allows GFR to increasew from 20 to 50 ml/min/1.73 m2

• Increases with time, reaching adult levels by 1 year of age

Urine Flow RateUrine Flow Rate

• Fetal kidneys excrete about 10 ml/kg/hr of urine with large Na content

• The 1st week of life there is an urine o.p., accounting for a 10% weight loss. Stimuli may be increased blood flow

• Voiding: 50% at 12 hrs. of life, 92% at 24 hrs and 99% at 48 hrs of life

FeNaFeNa

• In-utero: 15%

• 28 weeks: 5%

• 33 weeks: 3%

• Full term: 1-3%

PotassiumPotassium

• K levels in the NB are higher than in older babies (5.5-6.0 mEq/L)

• Renal CK in the NB = 9%

• Later in life = 15%

Acid-Base BalanceAcid-Base Balance

• Bicarbonate levels are lower in the newborn (19-21 mEq/L) due to endogenous acid production Bicarbonate reabsorption by the proximal

tubule– proton secretion in the collecting duct carbonic anhydrase activity

Web ReferenceWeb Reference

• http://physrev.physiology.org/cgi/content/full/79/4/1157#T1

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Nephrogenesis