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While the renal system is critical for maintaining homeostatic equilibrium within the body, it is also susceptible to various kinds of damage. Tubule dysfunction in particular contributes to acute renal injury and chronic kidney disease in millions of patients worldwide. Because current treatments are highly invasive and often unavailable, gaining a better understanding of the regenerative capacity of renal structures is vital. Although the effects of various types of acute damage have been previously studied, the ability of the excretory system to repair itself after dramatic tissue loss due to mechanical damage is less well characterized. A novel unilateral nephrectomy technique was developed to excise pronephric proximal tubules from Xenopus laevis tadpoles to study tubule repair after injury. Immunohistochemical detection of protein expression and renal uptake assays demonstrated that X. laevis larvae have the capacity to regenerate functional proximal tubules following resection. We have validated the renal identity of the restored tubules and demonstrated their ability to functional normally providing the first evidence of regeneration of renal tissue in an amphibian system. Importantly, this tubule restoration occurs by means of a process involving an early apoptotic event and the biphasic expression of the matrix metalloproteinase, Xmmp-9.
Figure 1. Proximal tubules are successfully excised during partial nephrectomy in NF stage 37/38 Xenopus laevis tadpoles. A: The 3G8-positive tissue was often partially (A) or completely ablated (B; left side) at 1 day postnephrectomy (dpn; 315 tadpoles), but occurred in the expected (phenotypically normal) pattern in the vast majority of damage control tadpoles (C; 52 tadpoles) and all unoperated sibling control tadpoles (D; 291 tadpoles). E: Damage in adjacent tissues was also examined by means of gene expression at 1 dpn. Normal expression of the alpha 5 subunit of Na, K-ATPase (E; 22 tadpoles), nephrin (F; 51 tadpoles), and 12/101 (G; 58 tadpoles) are retained in the pronephric duct, glomus, and skeletal muscle, respectively, on the operated side of the majority of nephrectomized tadpoles (H). A,C: All whole-mount photographs are lateral views of representative tadpoles, with anterior to the left. Black circles highlight the renal area. Black arrow marks the pronephric duct. White arrow indicates somitic muscle and white arrowhead points to hypaxial muscle. Photograph of 10 -μm section of paraffin-embedded/eosin counter-stained 1 dpn tadpole (B), with 3G8-positive proximal tubules (broken white outlines) present on the unoperated contralateral side but absent on the operated side. Scale bar = 100 μm; c, nephric coelom; g, gut; m, melanocytes (black arrowheads); n, notochord. Graph (H) illustrates the proportion of nephrectomized tadpoles with phenotypically normal, partially ablated, and completely ablated gene expression (in parentheses) in several tissues on the operated side.
Figure 2. Xenopus laevis tadpoles are able to regenerate proximal tubules 21 days after partial nephrectomy. A: The majority of nephrectomized tadpoles showed partial or no 3G8 expression at 1 day postnephrectomy (dpn). At 21 dpn, significantly more tadpoles expressed 3G8 normally in coiled proximal tubules on the operated side. Conversely, significantly fewer 21 dpn tadpoles displayed partial 3G8 expression. The proportion of 21 dpn tadpoles with no 3G8-positive ipsilateral tissue, is comparable to the percentage of the 1 dpn population with completely excised 3G8-positive tubules. A total of 315 animals was assayed at 1 dpn. A total of 458 animals was assayed at 21 dpn. Error bars indicate standard error among 19 replicates/group. Asterisks denote two-tailed Student's t-test between 1 dpn and 21 dpn groups: *P = 0.03; **P = 0.0001. B,C: All 21 dpn tadpoles with normal ipsilateral 3G8 expression in regenerated kidneys, displayed gene expression and tubule morphology similar to those observed in the unoperated sibling control tadpoles (compare B and C). Photographs are magnified lateral views of proximal tubules in representative tadpoles, with anterior to the left.
Figure 3. Regenerated pronephric proximal tubules are functional 3 weeks after excision. A: All unoperated control kidneys (115 animals) were able to reabsorb fluorescently tagged bovine serum albumin (BSA) into proximal tubules (A,B), as confirmed by the expression of the proximal tubule-specific protein, 3G8 (C). D: Of the 109 nephrectomized tadpoles assayed, 10 individuals (9%) restored ipsilateral coiled structures capable of both reabsorbing fluorescently-tagged-BSA (D,E) and expressing 3G8 (F). G: Comparable coiled morphology, BSA re-uptake capability and 3G8 expression were also seen in all unoperated contralateral proximal tubules. Photographs are lateral views of proximal tubules in representative tadpoles, with anterior to the left in A, and to the right in G. BF, brightfield. Broken outlines indicate proximal tubules. Solid outlines indicate distal tubules.
Figure 4. Nephrectomized tadpoles have increased levels of apoptosis during the first 24 hr postsurgery. A: A significant increase in the number of nephrectomized tadpoles expressing active Caspase 3 protein (as compared to both damage controls and unoperated sibling controls) was observed throughout the first day postsurgery. This number decreased significantly by the second day, and remained at low levels throughout the remainder of the first week postsurgery. A total of 250 animals from each treatment group were assayed at each time-point examined. Error bars indicate standard error among 2 replicates/group. Asterisks denote single-factor analyses of variance tests among different groups at the same time-point: *P < 0.05; **P < 0.001. B: Photographs are lateral views (anterior to the left) of an unoperated sibling control (B), damage control (C), and nephrectomized tadpole (D), at 12 hr postsurgery. White circle indicates renal area with active Caspase 3-expressing cells.
Figure 5. Xenopus laevis tadpoles express Xmmp-9 in a biphasic pattern after unilateral nephrectomy. A: In situ hybridization analysis demonstrated that Xmmp-9 expression was dramatically up-regulated in both nephrectomized and damage control groups within 3 hr of injury. Significantly more nephrectomized tadpoles displayed this phenotype between 12 and 24 hr postsurgery. This number decreased by 2 days postnephrectomy (dpn), and remained low until 5 dpn, at which time a second phase of Xmmp-9 up-regulation began on the ipsilateral side of only nephrectomized tadpoles. This phase persisted through 7 dpn. A total of 250 animals from each treatment group were assayed at time-points examined. Error bars indicate standard error among three to five replicates/group. Asterisks denote single-factor ANOVA tests among different groups at the same time-point: *P = 0.05; **P < 0.005. B: Photographs are lateral views (anterior to the left) of unoperated sibling controls (B,E), damage controls (C,F) and nephrectomized tadpoles (D,G), at 12 hr (B) or 7 days (E) postsurgery. Black circles indicate renal area in each tadpole.
