New imaging tools to measure nephron number in vivo: opportunities for developmental nephrology.

The mammalian kidney is a complex organ, requiring the concerted function of up to millions of nephrons. The number of nephrons is constant after nephrogenesis during development, and nephron loss over a life span can lead to susceptibility to acute or chronic kidney disease. New technologies are under development to count individual nephrons in the kidney in vivo. This review outlines these technologies and highlights their relevance to studies of human renal development and disease.

A population of mitochondrion-rich cells in the pars recta of mouse kidney.

Following perfusion of adult mouse kidney with a solution of nitroblue tetrazolium (NBT), certain epithelial cells in the pars recta (S3) segments of proximal tubules react to form cytoplasmic deposits of blue diformazan particles. Such cells are characterized by dark cytoplasm, small and often elliptical nuclei, elaborate, process-bearing profiles, and abundant mitochondria. The atypical epithelial cells display the additional characteristic of immunoreactivity for a wide spectrum of antigens, including mesenchymal proteins such as vimentin. Though present in kidneys of untreated or sham-operated animals, they are particularly evident under experimental conditions such as unilateral ureteral obstruction (UUO), appearing in both contralateral and obstructed kidneys over the course of a week's duration, but disappearing from the obstructed kidney as it undergoes the profound atrophy attributable to deterioration of the population of its proximal tubules. The cells do not appear in neonatal kidneys, even those undergoing UUO, but begin to be recognizable soon after weaning (28 days). It is possible that diformazan-positive cells in the mouse S3 tubular segment constitute a resident population of cells that can replenish or augment the tubule. Although somewhat similar cells, with dark cytoplasm and vimentin expression, have been described in human, rat, and transgenic mouse kidney (Smeets et al. in J Pathol 229: 645-659, 2013; Berger et al. in Proc Natl Acad Sci U S A 111: 1533-1538, 2014), those cells-known as "scattered tubule cells" or "proximal tubule rare cells"- differ from the S3-specific cells in that they are present throughout the entire proximal tubule, often lack a brush border, and have only a few mitochondria.

Glomerulotubular disconnection in neonatal mice after relief of partial ureteral obstruction.

Ureteropelvic junction obstruction is a common cause of congenital obstructive nephropathy. To study the pathogenesis of nephropathy, a variable-partial, complete or a sham unilateral ureteral obstruction (UUO) was produced in mice within 2 days of birth. The obstruction was released in some animals at 7 days and kidneys harvested at 7-42 days of age for histologic and morphometric study. Renal parenchymal growth was stunted by partial UUO with the impairment proportional to the duration and severity of obstruction. Proximal tubule apoptosis and glomerulotubular disconnection led to nephron loss. Relief of partial UUO arrested glomerulotubular disconnection, resolved tubule atrophy, and interstitial fibrosis with remodeling of the renal architecture. Relief of severe UUO did not result in recovery. Compensatory growth of the contralateral kidney depended on the severity of obstruction. Our studies indicate that relief of moderate UUO will minimize nephron loss. Application of this technique to mutant mice will help develop future therapies to enhance nephron recovery.

Specific molecular targeting of renal injury in obstructive nephropathy.

Progression of most renal disease involves tubulointerstitial injury, characterized by tubular atrophy, inflammatory cell infiltration, and interstitial fibrosis. Transforming growth factor-beta1 is central in this process. As reported by Moon et al., molecular targeting of the transforming growth factor-beta1 signaling pathway can markedly suppress renal injury resulting from unilateral ureteral obstruction, an established model of obstructive nephropathy. Specific kinase inhibitors are promising therapeutic agents to slow or attenuate progressive renal fibrosis.

Osteopontin regulates renal apoptosis and interstitial fibrosis in neonatal chronic unilateral ureteral obstruction.

Congenital obstructive nephropathy is a major cause of renal insufficiency in children. Osteopontin (OPN) is a phosphoprotein produced by the kidney that mediates cell adhesion and migration. We investigated the role of OPN in the renal response to unilateral ureteral obstruction (UUO) in neonatal mice. OPN null mutant (-/-) and wild-type (+/+) mice were subjected to sham operation or UUO within the first 2 days of life. At 7 and 21 days of age, fibroblasts (fibroblast-specific protein (FSP)-1), myofibroblasts (alpha-smooth muscle actin (SMA)), and macrophages (F4/80) were identified by immunohistochemical staining. Apoptotic cells were detected by terminal deoxy transferase uridine triphosphate nick end-labeling technique and interstitial collagen by Masson trichrome or picrosirius red stain. Compared to sham-operated or contralateral kidneys, obstructed kidneys showed increases in all parameters by 7 days, with further increases by 21 days. After 21 days UUO, there was an increase in tubular and interstitial apoptosis in OPN -/- mice as compared to +/+ animals (P<0.05). However, FSP-1- and alpha-SMA-positive cells and collagen in the obstructed kidney were decreased in OPN -/- compared to +/+ mice (P<0.05), whereas the interstitial macrophage population did not differ between groups. We conclude that OPN plays a significant role in the recruitment and activation of interstitial fibroblasts to myofibroblasts in the progression of interstitial fibrosis in the developing hydronephrotic kidney. However, OPN also suppresses apoptosis. Future approaches to limit the progression of obstructive nephropathy in the developing kidney will require targeting of specific renal compartments.

Distinct roles of Mac-1 and its counter-receptors in neonatal obstructive nephropathy.

Urinary tract obstruction during renal development leads to tubular atrophy and interstitial fibrosis. Inflammatory macrophages are crucial in this process, and beta2-integrins play a major role in leukocyte recruitment. We investigated the role of beta2-integrins and their major counter-receptors (intercellular adhesion molecule-1 (ICAM-1), receptor for advanced glycation endproducts (RAGE), junctional adhesion molecule (JAM)-C) in obstructive nephropathy in neonatal mice. Two-day-old beta2-integrin-deficient mice (Mac-1-/- and LFA-1-/-(deficient for leukocyte function-associated antigen-1)) and wild-type mice (C57BL/6) underwent unilateral ureteral obstruction (UUO) or sham operation. After 1, 5 or 12 days of obstruction, renal macrophage infiltration and tubulointerstitial damage were quantitated. Tissue abundance of Mac-1 and its ligands ICAM-1, RAGE and JAM-C was examined by Western blot and immunoprecipitation. Deficiency of either integrin was associated with reduced early macrophage invasion into the obstructed kidney. After 12 days of UUO, macrophage infiltration and tubulointerstitial injury were reduced only in Mac-1-/- but not in LFA-1-/- mice. Besides ICAM-1, an upregulation of two novel Mac-1 ligands, RAGE and JAM-C were observed, however, with distinct time courses. We conclude that beta2-integrins mediate macrophage infiltration in UUO. Mac-1 is the predominant leukocyte integrin involved in leukocyte recruitment after obstruction. ICAM-1 and its new ligands RAGE and JAM-C are sequentially activated in UUO. Blocking of Mac-1 and its ligands may confer synergistic renoprotective effects in neonatal obstructive nephropathy.

Renal apoptosis parallels ceramide content after prolonged ureteral obstruction in the neonatal rat.

Obstructive nephropathy, the primary cause of renal insufficiency in infants, is characterized by progressive renal apoptosis. Ceramide is a sphingolipid known to stimulate apoptosis in the kidney. We investigated the effects of unilateral ureteral obstruction (UUO) on endogenous renal ceramide content and apoptosis in neonatal and adult rats. Animals were subjected to UUO or sham operation on the first day of life and were studied 3-28 days later. Adult rats were similarly treated and then studied 3 or 14 days later. In additional neonatal rats, the obstruction was removed after 5 days, with study at 14 or 28 days. Renal ceramide content was measured by diacylglycerol kinase assay, and apoptosis was determined by the terminal deoxynucleotidyl transferase dUTP nick-end-labeling technique. Renal ceramide content was 50-fold higher in the 3-day neonatal compared with the adult kidney and 10-fold higher in the 7-day neonatal compared with the adult kidney, but there was no additional effect of UUO on ceramide content at these ages. However, after 14 or 28 days UUO in the neonate, renal ceramide was elevated compared with sham or intact opposite kidneys, and renal apoptosis was directly related to ceramide content (r = 0.99, P < 0.001). Moreover, renal ceramide was reduced by relief of obstruction (P < 0.05). There was less apoptosis in the obstructed kidney of the adult than the neonate, and UUO had no effect on ceramide content at 14 days in the adult. We conclude that prolonged UUO (at least 14 days duration) increases endogenous renal ceramide in the neonatal but not the adult rat. It is likely that this contributes to the prolonged renal apoptotic response of the neonatal obstructed kidney.

The moth and the aspen tree: sodium in early postnatal development.

Over the past 25 years, our perception of the neonatal kidney has changed markedly from its being a "limited" organ compared with that of the adult to being extraordinarily well adapted in its role in maintaining homeostasis and making possible the rapid somatic growth necessary during this critical period of life. The present review focuses on the physiologic adaptations by the neonatal kidney in the maintenance of a positive sodium balance, which is necessary for normal growth not only in mammals but also in moths. There is a fine interplay between the developing brain, heart, thyroid, adrenals, and sympathetic nervous system, all converging on the kidney to conserve sodium, which is limited in the diet. The renin-angiotensin system plays a central role in this response and is balanced by developmental changes in the renal response to atrial natriuretic peptide, all of which contribute to sodium conservation. Over the next 25 years, advances in molecular genetics will doubtless elucidate many more facets of the mechanisms underlying neonatal sodium homeostasis. This will be particularly important as the survival of ever smaller preterm infants improves steadily.

Unilateral ureteral obstruction in neonatal rats leads to renal insufficiency in adulthood.

BACKGROUND: Although unilateral ureteropelvic junction obstruction is the most common cause of congenital obstructive nephropathy in infants and children, management remains controversial, and follow-up after pyeloplasty is generally limited to the pediatric ages. We have developed a model of temporary unilateral ureteral obstruction (UUO) in the neonatal rat: One month following the relief of five-day UUO, the glomerular filtration rate (GFR) of the postobstructed kidney was normal despite a 40% reduction in the number of glomeruli and residual vascular, glomerular, tubular, and interstitial injury. METHODS: To determine whether hyperfiltration and residual injury of remaining nephrons leads to progression of renal insufficiency in later life, 31 rats were sham operated or subjected to left UUO at one day of age, with relief of UUO five days later, and were studied at one year of age. GFR was measured by inulin clearance, and the number of glomeruli, tubular atrophy, glomerular sclerosis, and interstitial fibrosis were measured by histomorphometry in sham, obstructed (UUO), and intact opposite kidneys. Intrarenal macrophages and alpha-smooth muscle actin were identified by immunohistochemistry. RESULTS: Despite relief of UUO, ultimate growth of the postobstructed kidney was impaired. The number of glomeruli was reduced by 40%, and GFR was decreased by 80%. However, despite significant compensatory growth of the opposite kidney, there was no compensatory increase in GFR, and proteinuria was increased. Moreover, glomerular sclerosis, tubular atrophy, macrophage infiltration, and interstitial fibrosis were significantly increased not only in the postobstructed kidney, but also in the opposite kidney. CONCLUSIONS: Although GFR is initially maintained following relief of five-day UUO in the neonatal rat, there is eventual profound loss of function of the postobstructed and opposite kidneys because of progressive tubulointerstitial and glomerular damage. These findings suggest that despite normal postoperative GFR in infancy, children undergoing pyeloplasty for ureteropelvic junction obstruction should be followed into adulthood. Elucidation of the cellular response to temporary UUO may lead to improved methods to assess renal growth, injury, and functional reserve in patients with congenital obstructive nephropathy.

Chronic ureteral obstruction in the rat suppresses renal tubular Bcl-2 and stimulates apoptosis.

Unilateral ureteral obstruction (UUO) results in widespread tubular apoptosis in obstructed kidneys of both adults and neonates. The oncoprotein bcl-2 inhibits many forms of apoptosis, whereas the related protein bax promotes apoptosis. To evaluate the interaction of bcl-2, bax, and apoptosis in the renal response to UUO, adult and neonatal rats were subjected to UUO or sham operation, and kidneys were harvested 14 days later. Apoptotic cells were identified by the Tunel technique, and the distribution of bcl-2 and bax was determined by immunochemistry. In both adults and neonates, tubular and interstitial apoptosis was present in the obstructed kidney, but not in intact kidneys. In both adults and neonates, there was diffuse tubular bcl-2 and bax staining of sham-operated and intact kidneys. While bcl-2 was increased in scattered nonapoptotic tubules of the obstructed kidney, there was minimal staining of dilated apoptotic tubules. These results are consistent with the premise that bcl-2 normally suppresses renal tubular apoptosis. The distribution of bax staining in tubules of the obstructed kidney overlapped that of bcl-2. We conclude that chronic UUO inhibits bcl-2 expression in selected tubules of the obstructed kidney which contributes to activation of apoptosis and progressive renal damage in either neonatal or adult kidneys. Dysregulation of apoptosis may be a response to renal injury similar to that underlying the development of cystic kidney disease or renal dysplasia.

Angiotensin stimulates TGF-beta1 and clusterin in the hydronephrotic neonatal rat kidney.

Unilateral ureteral obstruction (UUO) induces activation of the renin-angiotensin system and upregulation of transforming growth factor-beta1 (TGF-beta1; a cytokine modulating cellular adhesion and fibrogenesis) and clusterin (a glycoprotein produced in response to cellular injury). This study was designed to examine the regulation of renal TGF-beta1 and clusterin by ANG II in the neonatal rat. Animals were subjected to UUO in the first 2 days of life, and renal TGF-beta1 and clusterin mRNA were measured 3 days later. Rats were divided into treatment groups receiving saline vehicle, ANG, losartan (AT(1) receptor inhibitor), or PD-123319 (AT(2) receptor inhibitor). ANG stimulated renal TGF-beta1 expression via AT(1) receptors, a response similar to that in the adult. In contrast, clusterin expression was stimulated via AT(2) receptors, a response differing from that in the adult, in which ANG inhibits clusterin expression via AT(1) receptors. We speculate that the unique response of the neonatal hydronephrotic kidney to ANG II is due to the preponderance of AT(2) receptors in the developing kidney.

Renal tubulointerstitial injury from ureteral obstruction in the neonatal rat is attenuated by IGF-1.

BACKGROUND: The administration of insulin-like growth factor-1 (IGF-1) has been shown to ameliorate the renal injury resulting from ischemic acute renal failure. As there are a number of similarities between acute renal failure and obstructive nephropathy, we examined the effects of IGF-1 on the renal cellular response to unilateral ureteral obstruction (UUO) in the neonatal rat. METHODS: Forty-five rats were subjected to UUO or sham operation within the first 48 hours of life and received IGF-1 (2 mg/kg/day) or saline for the following three or seven days, after which kidneys were removed for study by morphometry and immunohistochemistry. To determine the effects of UUO on endogenous expression of IGF-1 and its receptor, six additional rats were subjected to UUO or sham operation, and mRNA was measured by solution hybridization. RESULTS: There was no effect of seven days of UUO on the renal expression of endogenous IGF-1 or its receptor. Moreover, seven days of exogenous IGF-1 did not improve the suppression of nephrogenesis, the delay in glomerular maturation, or the reduction in tubular proliferation induced by ipsilateral UUO. However, in the obstructed kidney, IGF-1 reduced tubular expression of vimentin, apoptosis, and tubular atrophy by 38 to 50% (P < 0.05). In addition, IGF-1 also decreased renal interstitial collagen deposition in the obstructed kidney by 44% (P < 0.05). Following three days of UUO, the administration of IGF-1 also reduced tubular apoptosis (P < 0.05), but did not alter tubular proliferation. CONCLUSIONS: IGF-1 has a profound salutary effect on the tubular and interstitial response to UUO in early development, without affecting glomerular injury or development. These results suggest that IGF-1 may have therapeutic potential in the management of congenital obstructive nephropathy.

Obstructive nephropathy: lessons from cystic kidney disease.

Obstructive nephropathy is one of the most important causes of renal failure in infants and children, while polycystic kidney disease (PKD) is a major cause of renal failure in the adult population. This review summarizes the evidence that there may be a number of mechanisms common to the pathophysiology of both conditions. In animal models of obstructive nephropathy and PKD, the renal tubular expression of epidermal growth factor is suppressed, and expression of clusterin is increased, both of which suggest arrested maturation or dedifferentiation of the tubular cell. There is a marked increase in apoptosis of epithelial cells in dilated tubules, associated with an increase in apoptotic stimuli. The renin-angiotensin system is activated in both obstructive nephropathy and PKD, which may contribute to tubular atrophy and interstitial fibrosis, which characterize the progression of both conditions. Focal cystic dilatation of the tubule is found in obstructive nephropathy, while tubular obstruction is present in cystic kidney disease. It is therefore likely that elucidation of the effects of mechanical stretch on renal tubular epithelial cells will contribute to our understanding of both conditions.

Apoptosis parallels ceramide content in the developing rat kidney.

Ceramide is emerging as an important hydrophobic sphingolipid involved in cell differentiation and apoptosis. Since apoptosis plays a significant role in cellular remodeling during renal morphogenesis, we measured ceramide content and apoptosis in the fetal (18 days gestation), neonatal (3, 7, and 14 days postnatal), and adult rat kidney. In addition, to determine whether developmental changes in ceramide content are tissue-specific, we compared renal ceramide content with that in lung and liver. Ceramide was measured by the diacylglycerol kinase assay, and apoptosis was determined by the TUNEL technique. Renal ceramide content fell over 100-fold from the fetus to the 7th postnatal day. Renal apoptosis paralleled ceramide content, with a greater than 300-fold decrease in apoptosis from fetal to adult life. Ceramide content of the lung and liver was significantly less than that of the kidney, and changed less with maturation. We conclude that maturational changes in ceramide content are tissue-specific, and that the high rate of apoptosis in the developing kidney may be related to the elevated ceramide content.

EGF improves recovery following relief of unilateral ureteral obstruction in the neonatal rat.

PURPOSE: Renal epidermal growth factor (EGF) is suppressed by unilateral ureteral obstruction (UUO), and we reported previously that exogenous EGF attenuates renal injury due to UUO in the neonatal rat. In this study, we wished to determine whether administration of epidermal growth factor (EGF) improves long-term renal cellular recovery after relief of obstruction. MATERIALS AND METHODS: One ureter of 1 day-old rats was occluded or sham-operated, and rats received daily injections of EGF, 0.1 mg./kg., or saline for the following 7 days. Five days following UUO, the obstruction was removed. Kidneys were removed 28 days following release of UUO or sham operation, and processed for histomorphometry and immunohistochemistry. RESULTS: Kidney weight and the number of glomeruli were reduced in the postobstructed kidney regardless of administration of EGF. However, EGF reduced tubular vimentin by 36% and clusterin expression by 70% (markers of tubular injury), and decreased tubular atrophy by 50% in the postobstructed kidney compared with saline-treated rats. EGF also reduced interstitial alpha-smooth muscle actin and interstitial collagen deposition by 50% in the postobstructed kidney. CONCLUSIONS: Short-term administration of EGF markedly attenuates both tubular and interstitial injury one month following the release of UUO in the neonatal rat. This suggests therapeutic potential for targeted delivery of growth factors to optimize recovery after release of urinary tract obstruction.

Molecular and cellular pathophysiology of obstructive nephropathy.

Congenital obstructive nephropathy remains one of the most-important causes of renal insufficiency in children. This review focuses on the unique interactions that result from urinary tract obstruction during the period of renal development in the neonatal rodent. Following unilateral ureteral obstruction (UUO), growth of the obstructed kidney is impaired and compensatory growth by the intact opposite kidney is related directly to the duration of obstruction. Development of the renal vasculature is delayed by UUO, and the activity of the intrarenal renin-angiotensin system is enhanced throughout the period of obstruction. Glomerular maturation is also delayed by UUO, and nephrogenesis is permanently impaired. The effects of UUO on the developing tubule are also profound, with a suppression of proliferation, stimulation of apoptosis, and the maintenance of an immature phenotype by tubular epithelial cells. Expression of tubular epidermal growth factor is suppressed and transforming growth factor-beta1 and clusterin are increased. Maturation of interstitial fibroblasts is delayed, with progression of tubular atrophy and interstitial fibrosis resulting in part from continued activation of the renin-angiotensin system and oxygen radicals. Future efforts to prevent the consequences of congenital urinary tract obstruction must account for the dual effects of obstruction: interference with normal renal development and progression of irreversible tubulointerstitial injury.

Renal cellular response to ureteral obstruction: role of maturation and angiotensin II.

Renal angiotensin II (ANG II) is increased as a result of unilateral ureteral obstruction (UUO), and angiotensin AT(2) receptors predominate over AT(1) receptors in the early postnatal period. To examine the renal cellular response to 3-day UUO in the neonatal and adult rat, AT(1) and AT(2) receptors were inhibited by losartan and PD-123319, respectively. Additional rats received exogenous ANG II, 0.5 mg. kg(-1). day(-1). Renal cellular proliferation and apoptosis were quantitated by proliferating cell nuclear antigen and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling technique, respectively. In the neonate, UUO reduced proliferation and increased tubular apoptosis. Losartan had no detectable cellular effect, whereas PD-123319 increased cellular proliferation and suppressed apoptosis, and exogenous ANG II stimulated apoptosis. In the adult, UUO increased cellular proliferation as well as apoptosis, whereas losartan, PD-123319, and exogenous ANG II did not alter the cellular response. In conclusion, UUO impairs renal growth in the neonate by reducing proliferation and stimulating apoptosis, at least in part through angiotensin AT(2) receptors. UUO stimulates both renal cellular proliferation and apoptosis in the adult, but these effects are independent of ANG II. We speculate that the unique early responses of the developing kidney to urinary tract obstruction are mediated by a highly activated renin-angiotensin system and preponderance of AT(2) receptors.