Epithelial abnormalities in intestine and kidney of the spontaneously hypertensive rat.

A variety of perturbations of calcium metabolism are reported to occur in the spontaneously hypertensive rat (SHR) compared to its genetic control the Wistar-Kyoto rat (WKY), including significant dysfunction of calcium handling by the proximal renal tubule of the SHR, resulting in impaired active calcium transport in the gut and an apparent renal calcium leak. We explored the intestinal and renal epithelia of 12- to 14-week-old SHR and WKY using electron microscopy. Biochemical comparisons of these transport epithelia included measurements of three vitamin D dependent cellular proteins and one structural protein: alkaline phosphatase, intestinal CaBP9K, renal CaBP28K, and villin expression. Electron microscopy demonstrated a patchy loss in microvilli in the SHR, accounting for approximately 10 to 15% of the total microvillar surface. In the kidney, morphological abnormalities were observed only in the proximal renal tubule. Again, there was patchy loss of microvilli from the brush border membrane. In SHR duodenal alkaline phosphatase activity was significantly reduced compared to the WKY (0.145 +/- 0.002 v 0.186 +/- 0.002 integrated extinction/min/micron 3 X 10(3) brush border (P less than .001). Duodenal CaBP9K and renal CaBP28K were significantly reduced in SHR compared to WKY. There were no differences in villin expression. These data are consistent with the previously characterized disturbances of active calcium transport in the intestine and inappropriate renal calcium leak in the SHR. While a possible link between these disturbances and hypertension remains to be determined, this study provides supportive evidence for a primary disturbance in cell calcium handling and transporting epithelia in this form of genetic hypertension.(ABSTRACT TRUNCATED AT 250 WORDS)

Ultrastructural and functional abnormalities of intestinal and renal epithelium in the SHR.

Intestinal calcium transport, renal tubular calcium reabsorption, and plasma 1.25 (OH)2 vitamin D3 (calcitriol) levels have all been reported to be diminished in the spontaneously hypertensive rat (SHR) compared with its genetic control the Wistar Kyoto rat (WKY). In the present study, absorptive duodenal and renal tubular epithelia of 12- to 14-week-old male SHR and WKY were examined by electron microscopy to determine whether such disturbances could be related to structural abnormalities. Patchy loss of microvilli in both duodenal and proximal tubular epithelia was observed in the SHR, whereas brush border membrane was entirely normal in the WKY. Irregular spaces were observed between the basal aspects of SHR intestinal epithelial cells and their basement membrane. In addition, the average height of duodenal and renal microvilli was reduced in the SHR. Two specific markers of the brush border membrane, alkaline phosphatase and villin, as well as the cytoplasmic vitamin-D dependent calcium-binding proteins, CaBP9K and CaBP28K were determined. Duodenal alkaline phosphatase activity was reduced in the SHR, compared with the WKY: 0.145 +/- 0.002 vs. 0.186 +/- 0.002 IE/min.microns 3 x 10(3) brush border, mean +/- SEM, N = 10 pairs, P less than 0.001. However, duodenal villin expression was not different from that of the WKY. Duodenal CaBP9K and renal CaBP28K content was diminished in the SHR: 21.0 +/- 0.80 vs. 29.9 +/- 2.19 micrograms/mg protein, N = 6 pairs, P less than 0.01 for duodenum, and 4.47 +/- 0.39 vs. 7.67 +/- 0.54 micrograms/mg protein, N = 6 pairs, P less than 0.001 for kidney. These data showing structural and functional abnormalities of intestinal and kidney cells in the SHR appear to reflect a disorder of transporting epithelia which may be either intrinsic or related to reduced circulating calcitriol.

Rapid (10-minute) stimulation of rat duodenal alkaline phosphatase activity by 1,25-dihydroxyvitamin D3.

Recent reports have suggested that the action of calcitriol is much more rapid than previously thought. It is thus possible that some actions do not depend on de novo protein synthesis. A precise microdensitometric technique has been used to characterize the time course of the intestinal brush border alkaline phosphatase (AP) response of rat duodenal villi to the administration of calcitriol as AP activity has been shown to be dependent on the vitamin D status of the animal. The technique enables AP activity to be determined in situ without tissue disruption. After ip administration of 200 ng calcitriol to vitamin D-deficient male Wistar AF rats, a biphasic AP response was observed with an early peak within 1 h (0.068 +/- 0.011 vs. 0.101 +/- 0.003 integrated extinction (IE) min.micron 3 X 10(3), P less than 0.05) and a second at between 6 and 8 h (0.088 +/- 0.005 vs. 0.172 +/- 0.003 IE/min.micron 3 X 10(3), P less than 0.001). In a further experiment, the early response to calcitriol was reexamined with observations at 0, 10, 30, 45, and 60 min after administration of either calcitriol or vehicle (n = 5 pairs per time point). AP activity was significantly increased in the calcitriol group compared with the vehicle-treated group as early as 10 min after administration (0.132 +/- 0.003 vs. 0.151 +/- 0.005 IE/min.micron 3 X 10(3), P less than 0.02) and reached a peak 45 min after administration at which time AP activity was equal to that found in normal vitamin D-replete animals (0.193 +/- 0.003 vs. 0.192 +/- 0.002 IE/min.micron (3) X 10(3), P greater than 0.5). The speed of this response indicates it to be unlikely to depend on de novo protein synthesis.

Effect of plasma levels of parathyroid hormone on NADPH pathways in kidney and liver.

NADPH available for mixed function oxidations (pathway 1) or biosynthetic processes (pathway 2) has been evaluated in different cells from rat liver and kidney. In addition, changes of the proportion of NADPH utilized in each pathway were demonstrated in the same cells from rats showing different circulating levels of parathyroid hormone (PTH). Quantitative levels of NADPH directed into each of these pathways have been measured and histologically located in sections from rat liver and kidney using quantitative cytochemistry and scanning and integrating microdensitometry. Centrilobular hepatocytes utilize the major amount of NADPH, either via pathway 1 or 2. Kidney cells utilize most NADPH via pathway 2, particularly in the distal part of the nephron. The cells of the pars recta have shown the highest capacity to utilize NADPH via pathway 1, which is about half that of centrilobular hepatocytes. In centrilobular cells, the presence of high plasma levels of PTH results in a significant increment of NADPH utilization either via pathway 1 or 2. In kidneys from rats showing high plasma levels of PTH, a selective increase in NADPH utilized via pathway 2 was observed in the distal convoluted tubule whereas a selective increase in NADPH utilized via pathway 1 was demonstrated in cells of the pars recta. These observations provide further information in the understanding of the physiology of kidney and liver cells.

In situ biochemical studies of intestinal alkaline phosphatase in normal and phosphate-depleted rats by microdensitometry.

A quantitative microdensitometric study has been designed to characterize in situ intestinal brush border-bound alkaline phosphatase of rat duodenal villosities. Intestinal slices were incubated with beta-glycerophosphate as substrate. Free phosphate liberated was precipitated in presence of a lead reagent as lead sulfide. The precipitate was quantified in situ by scanning and integrating microdensitometry. Kinetic parameters of the reaction were determined at 37 degrees C, pH 8.8, in the middle part of the villosities. Apparent Michaelis constant (Km) for beta-glycerophosphate was found to be 8.16 +/- 0.56 mM (mean +/- S.E.). Maximal enzyme activation was obtained at pH 8.5. Maximal inhibition of enzyme activity was observed in the presence of L-phenylalanine (30 mM) or theophylline (5 mM). Along the villosity axis, enzyme activity rose from the crypt up to the midportion of the villosity and finally decreased at the tip region. In phosphate-depleted rats, enzyme activity was increased in all portions of the villosity, with conservation of the same activity gradient. In this situation, kinetic analysis showed a marked decrease of Km, i.e. 4.56 +/- 0.39 mM (mean +/- S.E.) as compared to normal rats.