Growing Kidneys

Feb 16 2011 Published by under Nephrogenesis

H/T to @sciliz for the topic suggestion.

Normal Kidney Development

Pronephros and Mesonephros (Click for Source)

The first structure to develop in any vertebrate embryo, the pronephros, consists of a single filtering unit and tubule. In some species these structures do not attach to each other; a funnel-shaped structure with finger-like projections "waves" fluid from the site of filtration into the tubule. In some species the pronephros becomes the adult kidney, but in humans and "higher" vertebrates, the pronephros exists only briefly. It induces development of another structure, the mesonephros.

This mesonephros forms multiple filtration units and tubules capable of urinary function, albeit briefly in humans. These ducts become incorporated into the genitourinary system as a more complex metanephric kidney forms. In some animals, such as the zebrafish, the mesonephric kidney is the adult kidney.

Why Zebrafish?

Since the zebrafish kidney finishes at the mesonephric stage, it cannot be a perfect model for studying human kidneys; however, it offers many advantages over other models.

For example, the zebrafish embryo is transparent, making it easy to look at things without killing or cutting up the fry:

Zebrafish Embryonic Development (Link to YouTube Video)

Zebrafish breed rapidly and continuously with a generation time of 3 months.

Sequencing of the zebrafish genome facilitates creation of fish labelled with a variety of fluorescent markers, so specific structures can be studied in situ in live critters. Zhou et al labelled glomerular cells with a red marker and tubular structures with green fluorescent protein to make photos of normal nephrogenesis:

Mesonephrogenesis in cdh17::GFP/pod::mCherry double-transgenic zebrafish. A: schematic graph illustrating the transgene structures of pod::mCherry and cdh17::GFP. Tol2-L and Tol2-R are Tol2 transposon elements to facillite the transgenesis. B: lateral view of a 9 days postfertilization (dpf) larva. Podocytes in pronephric glomeruli (arrow) are marked with mCherry fluorescence (red) and pronephric tubules and ducts are labeled with GFP fluorescence (green). cdh17::GFP expression is also present in the intestinal epithelia, where the endogenous cdh17 is expressed (12). C: lateral view of a 14 dpf larva that developed the first pair of mesonephric glomeruli (arrowheads) with matured podocytes-expressing mCherry (red). D: ventral view of a 28 dpf juvenile. Multiple glomeruli (red) are visible at the anterior and medial portions of the mesonephric kidney with convoluted mesonephric tubules. E: ventral view of a 36 dpf juvenile. More mesonephric glomeruli and more convolutions of tubules developed in the anterior and medial regions. A few nephrons are present in between the rostral and medial regions as well as in the posterior region. F: ventral view of the whole mesonephric kidney in a 6-mo-old adult. ANDR, anterior nephron-dense region; MNSR, medial nephron-sparse region; MNDR, medial nephron-dense region; PNSR, posterior nephron-sparse region. Note symmetric groups of nephrons (arrowheads) in PNSR are segmentally distributed. Anterior to the left in all the panels.

Some background nephrogenesis can occur throughout the life of the zebrafish, but it slows down after 6 months of age. They then created a fish with the green marker linked to the zebrafish homolog of Wilm's Tumor 1, a marker of renal progenitor cells. Once again, podocin, a marker of mature glomeruli, drove the red marker. They then treated the fish with gentamicin, a nephrotoxic antibiotic, and showed that after kidney damage the zebrafish grew new nephrons:

Gentamicin-induced renal injury triggers de novo regeneration of mesonephric nephrons. A, A': injection of PBS does not increase mesonephrogenesis at 5 days postinjection (dpi). The green puncta are wt1b::GFP-expressing cells at the neck of matured nephrons. B, B': by 5 dpi of gentamicin, there is an increased number of developing nephrons. C, C': at 9 dpi of gentamicin, wt1b::GFP expression indicates the progression of these newly made nephrons. D, D': level of nephrogenesis is still elevated at 14 dpi of gentamicin. E: quantification of wt1b-GFP+ nephron numbers postgentamicin-induced renal injury. *P < 0.05, **P < 0.01.

So zebrafish can grow new nephrons with genetic markers similar to those of humans in response to kidney damage. Why can't people? And can we learn how zebrafish achieve this feat and make it happen with human cells? Can these nephrogenic cells be used to grow new nephrons in different zebrafish?Using a similar fluorescent tagging technique,  Diep et al transplanted progenitor cells from a zebrafish bearing one label to other fish with different labels and demonstrated that these cells would form new kidneys in the recipient fish. These cells worked with progenitor cells in the recipient fish to form the new units:

a, Overview of the transplantation assay. b, A primary transplanted fish at 18 d.p.t. with cdh17:EGFP+ donor-derived nephrons (arrow; inset, higher magnification view; scale bar, 0.5 mm). c, Average number of donor-derived nephrons over time (error bar, one standard deviation; n, total fish per time point). d, Head kidney of a recipient at 34 d.p.t. showing expansion of renal tissue caused by cdh17:mCherry+ donor-derived nephrons (arrow; scale bar, 0.5 mm). e, A cdh17:mCherry+ donor-derived nephron showing functional uptake of 40 kDa FITC-conjugated dextran (scale bar, 30 μm). f, Connection of donor-derived nephrons (cdh17:mCherry+) with the cdh17:EGFP+ recipient’s renal system (scale bar, 10 μm). g, A mosaic nephron arising from the co-injection of a mixture of cdh17:EGFP- and cdh17:mCherry-labelled nephron progenitors. h, Overview of the serial transplantation assay. i–k, Donor-derived nephrons (cdh17:EGFP+, arrows) in primary-, secondary- and tertiary-engrafted recipients (scale bar, 0.5 mm).

The Take-Home Message

First, even if you do not love kidneys and urine as much as I do, you must admit that these studies employ cool technology and generate amazing photos. Both of these articles include supplementary photos and videos as well; you should click on over to the web sites and play!

More important, Diep et al show that progenitor cells can be transplanted. Some dormant cells may remain in metanephric kidneys (like we humans have) that we can eventually learn to stimulate to regeneration. Someday we may be able to produce progenitor cells in vitro using stem cells, and then inject them into failing kidneys.

Finally, even with their differences, zebrafish can teach us important things about the development of organs, as well as being cute and hardy pets for our tanks.

Articles:

Zhou et al: Characterization of mesonephric development and regeneration using transgenic zebrafish. Am J Physiol Renal Physiol 299:F1040-F1047, 2010.  doi:10.1152/ajprenal.00394.2010

Diep et al: Identification of adult nephron progenitors capable of kidney regeneration in zebrafish. Nature 470:95-101, 2011.  doi:10.1038/nature09669

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