To determine the timing and location of renal cell regeneration after ischemic injury to the kidney and to assess whether exogenous epidermal growth factor (EGF) enhances this regenerative repair process to accelerate recovery of renal function, experiments were undertaken in rats undergoing 30 min of bilateral renal artery clamp ischemia followed by reperfusion for varying time intervals. Renal cell regeneration, as reflected by incorporation of radiolabeled thymidine within the kidney, began between 24 to 48 h and reached a peak at 72 h after renal ischemia. As demonstrated by histoautoradiography, renal thymidine incorporation was essentially confined to tubule cells. After the rats were killed by decapitation and exsanguination, the kidneys were quickly removed and cut longitudinally. Portions (100 mg) of the kidney were placed in cryogenic tubes and snap-frozen in liquid nitrogen. Samples were stored at -20 to -70'C for a maximum of 6 d before processing. Tissue samples were homogenized in ice-cold distilled water using a tissumizer (Tekmar Co., Cincinnati, OH). DNA was purified from 1.0 ml of homogenate. To obtain a complete time course, some animals were allowed to recover for 7 d after renal ischemia and blood samples were then obtained daily for BUN or serum creatinine measurements. Radioactivity was measured on 0.5 ml of sample in 20 ml of Safety-Solve (Research Products International Corp., Mt. Prospect, IL) in a scintillation counter (model 9000; Beckman Instruments, Inc., Fullerton, CA). Counting efficiency was estimated by an internal standard. Serum samples were counted to assure consistent intraperitoneal absorption of the [3H]thymidine. Samples of the DNA extract were counted and corrected for sample DNA content.To localize and identify the cells incorporating the radiolabeled thymidine within the kidney, histoautoradiography of kidney samples was also accomplished using methods previously published from this laboratory (5). Kidneys were perfused in vivo with 2% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.2). Tissue sections from kidneys were placed in 4% formaldehyde, 1% glutaraldehyde, 100 mM phosphate buffer (7.2). Specimens were dehydrated and embedded in methacrylate. Tissue sections (4 ,m) were dipped in NBT-2 nuclear emulsion (Eastman Kodak Co., Rochester, NY) and stored in the dark at 4°C for 21 d. The sections were developed and counterstained with Lee's methylene blue-basic fuchsin. For quantitative evaluation, a computerized operator-interactive system was used as described previously (5). Sections were examined at 176 magnification, and 10-15 fields were counted in the cortex and outer and inner stripes of the outer medulla in each section for an individual animal. The cell type of the labeled cells was identified as either tubular or interstitial.For the experiments that used EGF, rats were administered EGF (20 ,ug) subcutaneously 1-1.5 h after surgery. This dose of EGF is substantially lower than those employed in previously reported studies that administered EGF...

Repair and recovery of ischemic or nephrotoxic acute renal failure (ARF) are dependent upon renal tubule cell regeneration. Because epidermal growth factor (EGF) is a potent growth promoter to renal tubule cells, experiments were undertaken to assess the effects of exogenous administration of EGF during the recovery phase of HgCl2-induced ARF. Rats were administered HgCl2 (5 mg/kg sc), and [3H]thymidine incorporation into renal tissue and blood urea nitrogen (BUN) and serum creatinine concentrations were measured at various times after toxin administration. EGF (20 microgram) was administered subcutaneously 2 or 4 h after HgCl2 injection. Exogenous EGF resulted in greater levels of renal [3H]thymidine incorporation into renal proximal tubule cells compared with those observed in nontreated animals at several time points in the first 48 h after toxic injury. Morphometric analysis of histoautoradiograph sections of renal tissue demonstrated that greater than 96% of labeled cells were tubular in all examined sections. This EGF-related acceleration in DNA synthesis was associated with significantly lower peak BUN and serum creatinine levels, averaging 213 +/- 23 and 6.54 +/- 0.72 (SE) mg/dl, respectively, at 3 days in EGF-treated nephrotoxic rats compared with peak levels of 359 +/- 40 and 9.92 +/- 1.67 mg/dl (P less than 0.001, n = 7-16) at 5 days in non-EGF-treated nephrotoxic rats. EGF treatment also was associated with a return to near normal BUN and serum creatinine levels approximately 4 days earlier than that observed in non-EGF-treated animals. These findings demonstrate that exogenous EGF accelerates the repair process of the kidney after a severe toxic insult.

Kidney tubulogenesis is the initial step in renal organogenesis. The precise molecular determinants of this pattern formation are presently unknown, although soluble factors, such as growth factors, and insoluble factors, such as extracellular matrix molecules, most likely play fundamental roles in this process. To define the molecular determinants of renal proximal tubule morphogenesis, primary cultures of rabbit renal proximal tubule cells in hormonally defined, serum-free media were treated with transforming growth factor-beta 1 (TGF-beta 1), epidermal growth factor (EGF), and the retinoid, all trans-retinoic acid (RA), singly or in combination. Utilizing phase contrast and light and transmission electron microscopy, the simultaneous administration of TGF-beta 1 (10 ng/ml), EGF (1 nM), and RA (0.1 nM) transformed a confluent monolayer of renal proximal tubule cells within 5 to 6 days into three-dimensional cell aggregates containing lumens within the interior of the cell clusters. The lumens were bordered by tubule cells possessing a polarized epithelial cell phenotype with extensive microvilli formation and tight junctional complexes along the luminal border. All three factors were necessary and sufficient to induce this phenotypic transformation. Further studies demonstrated that RA promoted the deposition of the A and B1 chains of laminin, a cell attachment protein of the basement membrane, in a small subset of proximal tubule cells in culture, as deduced by indirect immunofluorescent microscopy. Additional studies demonstrated that soluble purified laminin fully substituted for RA in this system to promote renal tubulogenesis when combined with TGF-beta 1 and EGF. These results demonstrate that the growth factors, TGF-beta 1 and EGF, and the retinoid, RA, promote tubulogenesis in adult renal proximal tubule cells in tissue culture in a manner reminiscent of inductive embryonic kidney morphogenesis. These observations define a coordinated interplay between growth factors and retinoids to induce pattern formation and morphogenesis. Furthermore, the demonstration of RA-induced laminin deposition as a critical event in this morphogenic process identifies laminin as a possible target protein for RA to act as a morphogen.

Tissue engineering is a rapidly growing field in biotechnology. The use and packaging of synthetic materials, biologic compounds, and cellular components of specific tissues can be envisioned to replace physiologic function of diseased organs. Long-term ex vivo therapy for kidney failure has been achieved, so that the kidney may be the first solid organ in which tissue engineering concepts can produce an implantable device for long-term in vivo replacement therapy. To replace the kidney's excretory function, an implantable bioartificial kidney requires both a device to replace blood ultrafiltration performed by renal glomeruli and a device to replace transport regulatory function of the renal tubule. The initial concepts for these devices are just beginning to be considered and developed. (c) 1994 John Wiley & Sons, Inc.