Nephrogenesis

1. Nephrogenesis Maria E. Ferris, MD, MPH September 2001

3. Organogenesis • Most parenchymal epithelial organs follow a simple scheme of embryonic organogenesis. An epithelial sheet or tube thatis derived from one of the primordia, enters a process of sequentialbranching to generate a treelike structure. • In the kidney, the epithelial tube is to become the arborizingnephric duct-derived collecting duct system.

4. Nephrogenesis • A series of morphogeneticand differentiation events that starts with inductiveinteractions between 2 different primordial tissues and leads,in one of two mainstream processes: • formation of mesenchymalcondensations • formation of aggregates

5. Nephrogenic Stages • The kidney is derived from two different early embryonic tissue primordia: • The nephric duct  the mesonephric duct andcontinues through the Wolffian duct stage to the ureteric bud. • The nephrogenic cord, after inductive signaling with the pronephricduct-derived cells  the nephroi of mesonephros andmetanephros

6. Successive Bilateral Ontogenic Stages • Pronephros • Mesonephros • Metanephros

7. Pronephros • Occurs at the 3rd gestational week • About 7 tubules coalesce to form the pronephric duct

8. Mesonephros • 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

9. Metanephros • 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

13. Nephrogenesis • Occurs from 2 distinct embryologicalorigins • 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 inducedto branch, while the mesenchymal blastema is induced to enter thecritical process of mesenchyme-to-epithelium conversion or transition(MET).

14. 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]

15. 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 .

16. Mesenchyme-to-Epithelium Transition (MET) • These events are: • epithelial cellpolarization and • differentiationinto the highly specialized epithelial cell populations of thenephron

17. Signaling Molecules • Each step along the metanephrogenic pathwayis initiated and organized by signaling molecules that are locallysecreted polypeptides, encoded by different gene families and regulatedby transcription factors

18. 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).

19. Nephrogenesis • Proceeds from the medullato the outer cortex, directed by the ductal branching of the uretericbud-derived collecting tubule

20. 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

21. Nephrogenesis Model • Set up in the 1940’s, at the NIHdemonstrated in an organ system in vitro that • Kidney rudimentswhen removed at embryonic day 11 (E11mouse) follow an almostnormal developmental program in culture, • The isolated uretericbud cannot develop without contact to the metanephric mesenchyme,and • The isolated metanephrogenic mesenchyme can be inducedto go through the MET by a number of tissues, (embryonicspinal cord & the ureteric bud)

22. Regulation of Developmental Pathways • The nephrogenic mesenchyma and the ureteric budare regulated by • Transcription factors and proto-oncogenes, • Polypeptidegrowth factors acting as signaling molecules, and their receptors.

23. Modulation of Developmental Pathways • Modulated by cell adhesion molecule (CAM) complexes andtheir associations with the cytoskeleton, by extracellular matrix(ECM) glycoproteins and ECM receptor molecules such as the integrinfamily, and by ECM degrading proteases.

24. Growth Regulation • Proto-oncogenes that encode for receptor tyrosine kinasesare involved in mesenchymal (nephrogenic)-epithelial (ductopenic)interactions • Proto-oncogene encoded tyrosine or serine/threoninekinase, is the ureteric receptor for signalingmolecules secreted by the metanephrogenicmesenchyme • Proto-oncogenes regulategrowth and havethe potential to gain tumorigenesis after gene mutations (Wilms tumor).

25. Genetic 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.

26. 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]

27. 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.

28. Gene Mutations Influencing Nephrogenesis

29. 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

30. A. Growth Factors are Signaling Molecules • In addition to their mitotic (growth) action, not only mediate motogenic (migration) & morphogenic inductivesignals, but also those for cell differentiation (polarization),proliferation, and apoptosis. • The roles of the growth factorfamilies and their receptors, (each a multigenefamily) is very complex. • The best-characterizedgrowth factors pertinent to renal organogenesis are IGF-I andIGF-II, HGF, TGF- and FGF

31. 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

32. C. ECM and Cells Interact in Epithelial Morphogenesis • Most ECM molecules contain multiple binding domains that are recognized to interact with integrins, their glycoproteinshave signal transducing receptors & they mediate eventssuch as cell adhesion in embryonic cells • ECM molecules have morphoregulatory functions • Laminins transfer ECM signals to the cell • Integrin expression repertoire changes with nephrogenesis

33. 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

34. GENES THAT CONTROL NEHPROGENESIS • B. Signaling by Receptor Tyrosine Kinases • C-met • C-ret • C-ros

35. GENETIC ERRORS IN NEPHROGENESIS • Polycystic Kidney Disease • Dysruption of C-myc and bcl2 on ARPKD • Polycystin may participatein 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 matureepithelium may be a predisposition for oncogenesis

36. Newborn 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

37. Urine 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

38. FeNa • In-utero: 15% • 28 weeks: 5% • 33 weeks: 3% • Full term: 1-3%

39. Potassium • 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%

40. Acid-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

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