Morphometric and enzyme histochemical behaviour of the kidney of young rats before and after unilateral nephrectomy

Summary Four week old male Wistar rats were unilaterally nephrectomized, and one, two, four, fourteen, and twenty-eight days after the operation the remaining kidneys were removed for morphological examination—including measurement of diameters of the tubules—and for histochemical investigation of enzymatic activities in the nephrons. The following enzymes were examined: succinate DH, lactate DH, α-glycerophosphate DH, isocitrate DH, glucose-6-phosphate DH, cytochrome oxidase, adenosine triphosphatase, alkaline and acid phosphatases. In comparison with kidneys of control animals of the same age and sex, the hypertrophy of the remaining kidney was found to be, even in these young rats, entirely due to enlargement of preexisting nephrons. The measurements have indicated that the hypertrophy was most distinct between the second and fourth day after uninephrectomy. Four weeks after the operation the diameters of proximal and distal tubules were about 21% and 18% respectively bigger in the uninephrectomized animals than in the corresponding controls. This increase in size was accompanied by some changes in intensity of enzymatic staining reactions; after an intial diminishment, a graduate increase in activity of almost all the observed tubular enzymes was found; this seems to be the expression of the functional compensation by the remaining kidney. Paper dedicated to Professor Dr. Drs. h. c. W. Bargmann (Kiel) in honour of the 70th anniversary of his birthday.     

Excretion of water, sodium, and potassium during the compensatory renal hypertrophy in hypothyroid rats]

Renal compensatory hypertrophy is studied in age matched euthyroid and radiothyroidectomized female rats. 7 days after uninephrectomy, the hypertrophy of the remaining kidney is equally small in both groups. But 60 days after this operation, the hypothyroid animals show only a 12% increase in the wet weight of the remaining kidney whereas the euthyroid controls increase this weight by 21%. The excretion of water, Na and K are determined in the urine excreted in 5 h after a small water load. The results are related to 1 gram of kidney wet weight. These outputs increase in all animals after uninephrectomy. They are significantly higher in the hypothyroid rats than in the euthyroid controls as well before than 60 days after uninephrectomy. The reduction in tubular Na reabsorption found in the hypothyroid rat may account for the impairment of compensatory renal hypertrophy in hypothyroidism.     

Comparative characteristics of compensatory renal hypertrophy following unilateral nephrectomy in mature and old rats]

One of the kidneys was removed in rats from 35 to 900 g in weight; they were then sacrificed 2, 14, 30 and 60 days after the operation. The degree of compensatory hypertrophy of the remaining kidney varied greatly in rats of different age, without decreasing in old age, however. Sixty days after the operation the weight of the hypertrophic kidney in old rats was equal to 55--93% of the weight of both kidneys in control. Hypertrophy of the kidney at any age was accompanied by an increased proliferation of the tubular cells, particularly in their proximal portion. An increase in the size of renal bodies during kidney hypertrophy was characteristic of rats of any age. However, with the advance of age this process developed more rapidly and was stronger. At any periods of investigation the hypertrophic kidney in rats of any age contained a greater number (1 1/2--2 times more) of "open" renal bodies in comparison with the kidney of intact rats.     

Renal fibrosis in diabetic and aortic-constricted hypertensive rats

To assess if the renal damage observed in rats with diabetes and hypertension is due to hemodynamic or metabolic changes, a progressive aortic constriction between the two renal arteries has been done in streptozotocin-induced diabetic rats (constriction + diabetes group) and in nondiabetic rats (constriction group). This model allows us to study two kidneys subjected to different perfusion pressure (PP) in the same metabolic environment. One-month-old rats (100-120 g body wt) were subjected to the aortic constriction procedure. Three months after constriction, glomerular filtration rate and renal plasma flow were similar in both kidneys of the two groups. PP was greater in the kidney placed over the ligature [constriction high-pressure kidney (CH) or constriction + diabetic high-pressure kidney (DH)] than in the one placed below the ligature [constriction low pressure (CL) or constriction + diabetic low pressure (DL)]. Proteinuria was higher in the CH than in the CL kidneys (512 +/- 61 vs. 361 +/- 38 microg/30 min, respectively) and much higher in the DH kidney (770 +/- 106 microg/30 min). Renal fibrosis was measured in tissue sections stained with Syrius red using a computer-assisted image analysis system. DH and DL kidneys showed higher corpuscular cross-sectional and capillary tuft areas than the CH and CL ones. The DH kidney showed slight mesangial expansion and thickening of the capillary walls, which were more pronounced in the former. Most renal corpuscles from CH and DH groups were nearly normal in morphology appearance, and only in some instances a slight increment in mesangium was observed. Transforming growth factor-beta1 (TGF-beta1) immunostaining revealed that DH kidneys showed the highest glomerular expression. We concluded that 1) diabetic animals develop glomerular but not interstitial fibrosis to a greater extent than nondiabetic animals and that this lesion principally occurs in the hypertensive kidney (DH), and 2) increased TGF-beta expression is associated with diabetic renal damage.     

Changes in glomerulotubular dimensions, single nephron glomerular filtration rates and the renin-angiotensin system in hypothyroid rats

Glomerular diameters (GD) and lengths of attached proximal convoluted tubules (TPL) were measured in nephrons dissected from the superficial (S), intermediate and juxtamedullary (JM) cortex (7-15 each) of acid-macerated kidneys of weight-matched (E) euthyroid and (H) hypothyroid (2-6 months after radioiodine treatment or thyroidectomy) male Sherman-Wistar rats. Incoordination of growth in H rats was evident in a more marked retardation in kidney than in total body growth. A similar incoordination of microstructural growth was evident in maintenance or GD within normal limits with respect to body weight while attached TPL fell 23% on the average below control values relative to body weight. These changes affected the total nephron population uniformly. As a result, GD/TPL in all nephrons increased significantly (p less than 0.01), by 27% in S and by 29% in JM nephrons. The glomerulotubular dimensional imbalance was associated with a marked and uniformly distributed reduction in single nephron glomerular filtration rate (ferrocyanide method), by 36% in S and JM nephrons. Plasma renin activity fell within normal limits while plasma renin substrate was decreased to 56% of control values. These findings are construed as evidence that growth retardation in hypothyroid rats affects the parenchyma of the kidney (and perhaps other viscera) more than the vasculature.     

Enhancement of maternal and fetal nephrotoxicity of salicylate by zinc deficiency. Morphological, enzyme histochemical and immunohistochemical studies

An oral dose of 700 mg/kg salicylic acid was given to normal and Zn-deficient rats at day 16 of gestation. Maternal and fetal kidneys were studied at day 19 of gestation. Zn-deficiency did not cause any lesions detectable by semi-thin section light microscopy, electron microscopy, enzyme histochemistry and immunohistochemistry. Salicylate may lead only to small morphological, enzymatic and cytoskeletal defects in the maternal and fetal kidney. However, enzyme activities decreased in plasma membranes, mitochondria, lysosomes, endoplasmic reticulum and peroxisomes in all segments of the tubular apparatus when salicylate was given to Zn-deficient rats. Cytoskeletal proteins such as keratin in the glomerular cells and epithelial lining of the collecting ducts and vimentin in vascular endothelial cells of the maternal kidney were also affected. In addition, the epithelial cells of the collecting ducts, which were comparatively less damaged, accumulated high amounts of fat. In severe cases, the enzymatic and cytoskeletal lesions were accompanied by hematuria and tubular necroses including and collecting ducts in the renal papilla. In less severe cases reduced activities of brush border hydrolases were the only sign of disturbed renal function in maternal rats indicating that membrane alteration and loss of membrane-bound enzymes are the primary defects. In the fetal kidneys, mitotic activity of the cells of the nephron anlagen and collecting ducts was reduced and enzymatic and morphological differentiation were disturbed. As a consequence less mature nephrons and collecting ducts occurred.     

Maturation of glomerular size distribution profiles in domestic fowl (Gallus gallus)

Previous histological evaluations of chick kidneys indicated nephrons continue to develop from embryonic foci for up to 6 weeks after hatching. The present study was conducted using an in vivo alcian blue staining technique to quantify posthatch changes in glomerular numbers and sizes in female domestic fowl at 1, 3, 5, 9, 12, 21, and 30 weeks of age. Changes in glomerular size distributions reflect changes in the heterogeneous nephron populations of avian kidneys. Foci of embryonic tissue were observed at the periphery of renal lobules up to 12 weeks of age. Glomerular numbers increased from 69,800/kidney at 1 week to 586,000/kidney at 12 weeks, with no further significant increase up to 30 weeks (599,000/kidney). The increase in glomerular number per gram kidney weight remained constant as kidney mass increased up to 12 weeks of age, after which the number of glomeruli per gram kidney weight declined significantly as kidney size increased without further addition of new nephrons. Glomerular size distribution profiles were constructed using eleven circumference categories. The peak number of glomeruli fell within the 0.11-0.14 mm category at 1 and 3 weeks; within the 0.15-0.18 mm category at 5, 9, and 12 weeks; and within the 0.19-0.22 mm category at 21 and 30 weeks. One and 3-week-old chicks had no glomeruli within the largest (greater than or equal to 0.35 mm circumference) size categories, and 9-12-week-old birds had significantly fewer glomeruli in these categories than 21-30-week-old birds. These results demonstrate that posthatch renal maturation in domestic fowl involves the ongoing formation of new nephrons up to 12 weeks of age, with subsequent kidney growth (12-30 weeks of age) accomplished by enlargement of existing nephrons (nephron hypertrophy). The cumulative evidence indicates that nephrons destined to develop loops of Henle (mammalian-type) develop first, with shorter (reptilian-type) nephrons developing later as the kidneys enlarge.     

Sialic acid and neuraminidase activity in rat kidneys 6 months after uninephrectomy

Sialic acid and neuraminidase activity were determined in the cortex of the remnant kidneys of six uninephrectomized rats. As controls served either the kidneys removed at operation or age-matched kidneys from eight sham operated rats. Six months after uninephrectomy the kidneys became hypertrophied and their mean weight was about 40% higher than age-matched kidneys. Blood urea and creatinine and protein levels in 24-hr urine collections were significantly higher in the experimental animals as compared to those of the same animals before nephrectomy and to sham operated rats, indicating a marked impairment of kidney function. The mean concentration of sialic acid in the cortex of hypertrophied kidneys was not statistically different from either that of the removed or that of the age-matched kidneys. Neuraminidase activity expressed as either per gram fresh tissue or per milligram protein, was not different in the removed and in the hypertrophied kidney. The activity, however, in the latter was significantly lower than in the age-matched kidneys. Whether this finding can be associated with the impairment of kidney function in rats 6 months after uninephrectomy remains to be studied.     

Enhancement of maternal and fetal nephrotoxicity of salicylate by zinc deficiency

Summary An oral dose of 700 mg/kg salicylic acid was given to normal and Zn-deficient rats at day 16 of gestation. Maternal and fetal kidneys were studied at day 19 of gestation. Zn-deficiency did not cause any lesions detectable by semi-thin section light microscopy, electron microscopy, enzyme histochemistry and immunohistochemistry. Salicylate may lead only to small morphological, enzymatic and cytoskeletal defects in the maternal and fetal kidney. However, enzyme activities decreased in plasma membranes, mitochondria, lysosomes, endoplasmic reticulum and peroxisomes in all segments of the tubular apparatus when salicylate was given to Zn-deficient rats. Cytoskeletal proteins such as keratin in the glomerular cells and epithelial lining of the collecting ducts and vimentin in vascular endothelial cells of the maternal kidney were also affected. In addition, the epithelial cells of the collecting ducts, which were comparatively less damaged, accumulated high amounts of fat. In severe cases, the enzymatic and cytoskeletal lesions were aecompanied by hematuria and tubular necroses including the collecting ducts in the renal papilla. In less severe cases reduced activities of brush border hydrolases were the only sign of disturbed renal function in maternal rats indicating that membrane alteration and loss of membrane-bound enzymes are the primary defects. In the fetal kidneys, mitotic activity of the cells of the nephron anlagen and collecting ducts was reduced and enzymatic and morphological differentiation were disturbed. As a consequence less mature nephrons and collecting ducts occurred.Dedicated to Professor Zdenek Lojda on the occasion of his 60th birthdaySupported by the German Research Foundation (Sfb 174)     

Renal Primordia Activate Kidney Regenerative Events in a Rat Model of Progressive Renal Disease

New intervention tools for severely damaged kidneys are in great demand to provide patients with a valid alternative to whole organ replacement. For repairing or replacing injured tissues, emerging approaches focus on using stem and progenitor cells. Embryonic kidneys represent an interesting option because, when transplanted to sites such as the renal capsule of healthy animals, they originate new renal structures. Here, we studied whether metanephroi possess developmental capacity when transplanted under the kidney capsule of MWF male rats, a model of spontaneous nephropathy. We found that six weeks post-transplantation, renal primordia developed glomeruli and tubuli able to filter blood and to produce urine in cyst-like structures. Newly developed metanephroi were able to initiate a regenerative-like process in host renal tissues adjacent to the graft in MWF male rats as indicated by an increase in cell proliferation and vascular density, accompanied by mRNA and protein upregulation of VEGF, FGF2, HGF, IGF-1 and Pax-2. The expression of SMP30 and NCAM was induced in tubular cells. Oxidative stress and apoptosis markedly decreased. Our study shows that embryonic kidneys generate functional nephrons when transplanted into animals with severe renal disease and at the same time activate events at least partly mimicking those observed in kidney tissues during renal regeneration.     

Long-Term Responses to Loss of Renal Mass: Pathophysiological Aspects: The Influence of Age on the Response to Renal Parenchymal Loss

The effect of age on compensatory hypertrophy and functional adaptation to loss of 75 percent of renal mass was studied in canine puppies. In one group of animals the surgery was done between 1-5 days after birth and in another group, at two months of age. All animals were studied six weeks later. Shamoperated littermates served as controls. The newborn puppies in the experimental group were able to grow and maintain homeostasis as well as their controls, whereas the older experimental animals grew poorly and had significantly higher levels of plasma creatinine than their sham-operated counterparts (p < .05). The increase in mass of the remaining kidney was twice as much in the newborn as in the older dogs. Functional adaptation, as expressed by GFR, was nearly complete in the young, but reached only about 45 percent of controls in the older age group (p<.005). The intrarenal blood flow distribution was similar for experimental and control animals in both groups studied. There were, however, marked differences in the pattern of single glomerular perfusion rates: whereas in the older dogs the increase was confined to the deeper nephrons, in the newborn an increase occurred in all zones of the kidney. These studies demonstrate that compensation for massive loss of renal tissue is complete when the injury is sustained in the immediate postnatal period but only partial when it occurs later on in life. A loss in the adaptive capacity of the superficial nephrons appears to account for this age-related difference.     

Biochemical and Molecular Responses to Loss of Renal Mass: Atpase and Cyclic Nucleotides: Changes in Renal Cyclic Nucleotides as a Trigger to the Onset of Compensatory Renal Hypertrophy

In adult male Wistar rats contralateral nephrectomy was followed, within 10 minutes, by a nearly twofold rise of the content of cGMP in renal tissue. 20 and 40 minutes after contralateral nephrectomy cGMP fell to one half its control level to rise again to its normal level within 90 minutes. The initial rise of the concentration of cGMP was accompanied by a simultaneous fall of the concentration of cAMP by about 30 percent: the cAMP concentration remained 10-20 percent below control level for approximately two hours and rose again to its initial level after three hours. Cross-circulation of a nephrectomized rat with an intact animal led to a sharp increase of cGMP in the kidneys of the latter with a peak at 10 minutes after initiating cross-circulation and also to a fall of the cAMP concentration. When the same nephrectomized donor rat was subsequently cross-circulated with one, or even two, intact receiver animals, similar short-lasting changes of cyclic nucleotide concentrations were recorded in the kidneys of all the receivers. When a normal kidney was transplanted to the neck of a rat, subsequent removal of one of its own kidneys did not result in any change in cyclic nucleotide content in either the remaining or the transplanted kidney. The data are interpreted to indicate that renal tissue produces a factor inhibiting renal growth which counteracts a circulating humoral kidney growth stimulating factor of unknown origin. An initial rise of cGMP and a fall of cAMP may trigger the subsequent stimulation of protein synthesis responsible for hypertrophy.     

Modification of genital kidney nephrons for sperm transport in a plethodontid salamander,Eurycea longicauda

Salamanders possess kidneys with two distinct regions: a caudal pelvic portion and cranial genital portion. Nephrons of the pelvic region are responsible for urine formation and transport. Nephrons of the genital region transport sperm from testes to Wolffian ducts; however, nephrons of the genital region possess all the same functional regions found in pelvic kidney nephrons that are involved with urine formation and transport (renal corpuscles, proximal tubules, distal tubules, and collecting ducts). Morphological similarities between pelvic and genital regions stimulated past researchers to hypothesize that nephrons of genital kidneys possess dual function; that is, sperm transport and urine formation/transport. Considering size of glomeruli is directly related to the total amount of blood plasma filtered into the Bowman's space, we tested the hypothesis that nephrons of genital kidneys have reduced urine formation function by comparing glomerular size between nephrons of pelvic and genital kidney regions in Eurycea longicauda with general histological techniques. Light microscopy analysis revealed that glomeruli of pelvic kidneys were significantly larger than those measured from genital kidneys. Transmission electron microscopy analysis also revealed modifications in genital kidney nephrons when compared to pelvic kidney nephrons that suggested a decrease in urine formation function in genital kidneys. Such modifications included a decrease in basal and lateral plasma membrane folding in genital kidney proximal and distal tubules compared to that of pelvic kidney proximal and distal tubules. Genital kidney proximal tubules were also ciliated, which was not observed in pelvic kidney proximal tubules. In conclusion, although structurally similar at the histological level, it appears that nephrons of genital kidneys have decreased urine formation function based on glomerular size comparison and nephron ultrastructure.     

Long-Term Responses to Loss of Renal Mass: Glomerular Changes: Evidence of Induction of New Nephrons in Immature Kidneys Undergoing Hypertrophy

Unilateral nephrectomy performed during the first days or weeks of life may or may not induce the development of new nephrons in the remaining kidney. Such an increase has been reported to occur, as discussed in this review, in newborn rats and mice, but not in guinea pigs. These observations are consistent with other data suggesting different patterns of compensatory adaptation in young and in old animals.     

Renal glutathione homeostasis in compensatory renal growth

Glutathione homeostasis was investigated in unilaterally nephrectomized and sham-operated rats. Following twelve days of compensatory renal growth, it was found that the concentrations of glutathione and glutathione disulfide in representative samples of the entire remnant right kidney from the nephrectomized rats were similar to those found in corresponding samples of the right kidneys from the sham-operated rats. However, since the mass of the remnant right kidneys in the nephrectomized rats was greater than that of the right kidneys from the sham-operated rats, the absolute content of glutathione and glutathione disulfide was greater in the remnant right kidneys of the nephrectomized rats than in the right kidneys of the sham-operated rats. In general, the findings from the present study indicate that the absolute content of glutathione and glutathione disulfide in renal epithelial cells increases in proportion to the increase in mass that results from compensatory renal cellular hypertrophy.     

Influence of nutritional factors on the renotrophic action of ACTH in the uninephrectomized rat.

In the rat, renal compensatory hypertrophy (RCH) was apparent 48 h after uninephrectomy; it was significantly enhanced by long-acting beta1-24-corticotrophin (ACTH) when the animals had free access to food and a NaCl solution (9 g/l). In rats starved after uninephrectomy but drinking the NaCl solution freely, RCH was suppressed: the weights of the body, heart, liver, and solitary kidney were reduced. In similarly starved rats treated with ACTH, the weights of the heart and the solitary kidney were normal. RCH was also impaired in rats fed only a glucose solution (30 g/dl) after uninephrectomy, but it is restored by ACTH, which significantly increases the weight of the remaining kidney. This renotrophic action of ACTH may be related to hyperglycemia and, perhaps, elevated urinary K excretion, which occur in hyper-adrenocorticism and increase the work load of the nephron.     

Cysts in cultured fetal rat kidneys.

The formation of cysts has been studied in kidneys removed from day-15 and day-18 rat fetuses and cultured in a mixture of medium 199 (Morgan et al., '50) for periods of up to 15 days. Gas phases of 95% O2 and 5% CO2, and 95% air and 5% CO2, were employed, the latter being considered more satisfactory. In day-15 kidneys cysts formed from the ampullary portions of the collecting tubules after 2 days whereas cysts derived from nephrons were not seen until 9 days of culturing. The latter arose from developing juxtamedullary nephrons. In day-18 kidneys cysts from collecting tubules and nephrons were both present after 3 days of culturing. The latter, in this instance, originated mostly in immature midcortical nephrons, the juxtamedullary mephrons having undergone rapid degeneration. The tubular portion of the nephron seemed to be the primary site of dilatation. Under culture conditions cysts of nephrons thus formed from immature actively developing nephrons and not from those that were mature. Cysts associated with collecting tubules arose from the ampullary (terminal) portions of the latter in both day-15 and day-18 cultured kidneys. The study of cultured mammalian fetal kidneys can provide information on the nature and genesis of renal cysts. It is possible that the same technique also may be helpful for examining the effects of teratogens directly on the kidney.     

7-Hydroperoxycholesterol as a marker of oxidative stress in rat kidney induced by paraquat

The in vivo paraquat-induced oxidative stress in rat tissue was studied by analyzing cholesterol-derived hydroperoxide as an index of lipid peroxidation. Paraquat (10 mg/kg) was administered i.p. to rats. Rats were sacrificed and lung, liver, and kidney were collected 2, 24 h, and 5 d after paraquat injection. Lipids were extracted and analyzed by HPLC with post-column chemiluminescence. We found that two cholesterol-derived hydroperoxides, 7alpha-hydroperoxycholest-5-en-3beta-ol (7alpha-OOH) and 7beta-hydroperoxycholest-5-en-3beta-ol (7beta-OOH) were present in lungs of control animals (0.06 and 0.06 nmol/g, respectively), in livers (6.5 and 15.8 nmol/g, respectively) and in kidneys (3.7 and 8.9 nmol/g, respectively). In liver paraquat increased lipid peroxidation approximately by 60% over the levels of control animals only at 2 h after paraquat treatment. In kidney, augmented lipid peroxidation, 7alpha-OOH and 7beta-OOH (by 70% and 147%, respectively) above levels was found at 2 h after paraquat treatment. Interestingly, these increase remained in kidney of rats 5 d after a single dose of paraquat. In contrast, cholesterol-derived hydroperoxides were not affected in lung of paraquat dosed rats. This is the first report on 7alpha-OOH and 7beta-OOH accumulations in rat liver and kidney, and it seems to reflect greater oxidative stress in the pathology of kidney of rats treated with acute paraquat at low dose.     

Chronic Akt blockade aggravates pathological hypertrophy and inhibits physiological hypertrophy

The attenuation of adverse myocardial remodeling and pathological left ventricular (LV) hypertrophy is one of the hallmarks for improving the prognosis after myocardial infarction (MI). The protein kinase Akt plays a central role in regulating cardiac hypertrophy, but the in vivo effects of chronic pharmacological inhibition of Akt are unknown. We investigated the effect of chronic Akt blockade with deguelin on the development of pathological [MI and aortic banding (AB)] and physiological (controlled treadmill running) hypertrophy. Primary cardiomyocyte cultures were incubated with 10 μmol deguelin for 48 h, and Wistar rats were treated orally with deguelin (4.0 mg·kg(-1)·day(-1)) for 4 wk starting 1 day after the induction of MI or AB. Exercise-trained animals received deguelin for 4 wk during the training period. In vitro, we observed reduced phosphorylation of Akt and glycogen synthase kinase (GSK)-3β after an incubation with deguelin, whereas MAPK signaling was not significantly affected. In vivo, treatment with deguelin led to attenuated phosphorylation of Akt and GSK-3β 4 wk after MI. These animals showed significantly increased heart weights and impaired LV function with increased end-diastolic diameters (12.0 ± 0.3 vs. 11.1 ± 0.3 mm, P < 0.05), end-diastolic volumes (439 ± 8 vs. 388 ± 18 μl, P < 0.05), and cardiomyocyte sizes (+20%, P < 0.05) compared with MI animals receiving vehicle treatment. Furthermore, activation of Ca(2+)/calmodulin-dependent kinase II in deguelin-treated MI animals was increased compared with the vehicle-treated group. Four wk after AB, we observed an augmentation of pathological hypertrophy in the deguelin-treated group with a significant increase in heart weights and cardiomyocyte sizes (>20%, P < 0.05). In contrast, the development of physiological hypertrophy was inhibited by deguelin treatment in exercise-trained animals. In conclusion, chronic Akt blockade with deguelin aggravates adverse myocardial remodeling and antagonizes physiological hypertrophy.     

Immunohistological demonstration of pyruvate kinase isoenzyme type L in rat with monoclonal antibodies.

Four monoclonal antibodies (MAb) specific for the L-type isoenzyme of rat pyruvate kinase (L-PK) were produced and characterized. They detect at least two different epitopes of the isoenzyme, as shown in interference binding assay and Western blot analysis after peptide mapping. The MAb were used in immunohistology to demonstrate the L-PK isoenzyme in paraffin-embedded normal rat tissues. L-PK was found only in hepatocytes, kidney proximal tubules, islet cells of pancreas, and epithelial cells of the villi of small intestine. The content of L-PK in hepatocytes was often lower in the periportal areas as compared with the periveneous zone. In kidneys a clearcut difference in L-PK content and distribution existed between male and female rats. Male animals possessed more L-PK in the kidney cortex than females. The L-PK content in the inner cortical zone (straight proximal tubules) was higher than in the outer cortical zone (convoluted proximal tubules) in kidneys of males. In contrast, female rats displayed less L-PK in the inner than in the outer cortical zone of the kidneys. Only some of them exhibited the same amount of the isoenzyme in both parts of the kidney proximal tubules.