The urinary concentrating defect in the Gunn strain of rat. Role of bilirubin.

The role of high serum and tissue levels of unconjegated bilirubin in the pathogenesis of the impaired urinary concentrating ability was investigated in homozygous (jj) Gunn rats with the congenital absence of hepatic glucuronyl transferase. Continuous phototherapy with blue fluorescent lights at a wave length of 460 nm or oral cholestyramine feeding or both reduced serum levels of unconjugated hilirubin to levels consistently below 3.0 mg/100 ml for several weeks in both weanling and adult jj Gunn rats. The renal concentrating defect was already present in weanling jj Gunn rats by 21 days of age. In treated weanling jj animals, maximum concentrating ability and the concentration of urea and nonurea solutes in the papilla and medulla, determined after 24 h of fluid deprivation, were normal when compared to unaffected heterozygous (Jj) littermates. Solute-free water reabsorption which is reduced in jaundiced jj Gunn rats was restored to normal in treated weanling jj rats. The tissue concentration of unconjugated bilirubin was reduced throughout the papilla and inner and outer medulla in the treated jj rats in comparison with untreated jj littermates. The defect in urinary concentrating ability was only partially reversible and sometimes irreversible in adult jj rats, probably because of permanent renal parenchymal damage occurring secondary to massive crystalline deposits in the papilla and medulla. It is concluded that unconjugated bilirubin is directly involved in the pathogenesis of the concentrating defect in jaundiced jj Gunn rats.

Response of the distal tubule and cortical collecting duct to vasopressin in the rat.

Renal micropuncture observations in the rat suggest that the entire "distal tubule" (defined by the micropuncturist as that portion of the renal tubule extending between the macula densa and its first junction with another (renal tubule) may be responsive to vasopressin. However, this portion of the renal tubule contains two segments that are morphologically dissimilar. The "early" distal tubule is lined by epithelium characteristic of the distal convoluted tubule, while the "late" distal tubule is lined by epithelium characteristic of the cortical collecting duct. Thus, the present study was initiated to identify the most proximal site of action of vasopressin in the distal renal tubule. A water diuresis was established in rats with hereditary hypothalamic diabetes insipidus. In one-half of the animals the diuresis was interupted by an i.v. infusion of exogenous vasopressin. Morphological preservation of the kidneys was initiated after induction of vasopressin-induced antidiuresis or during maximum water diuresis. Cell swelling and dilatation of intercellular spaces, morphological findings indicative of vasopressin responsiveness, were observed in the cortical collecting duct including the late segment of the distal tubule, a segment that has also been described by morphologists as the initial collecting tubule. Morphological evidence of vasopressin-responsiveness was not observed in the early distal tubule (distal convoluted tubule). Additional morphological studies in Wistar, Long-Evans, and Sprague-Dawley rats demonstrated a marked difference in the random availability of distal convoluted tubules versus initial collecting tubules potentially available for micropuncture just beneath the renal capsule. The results suggest that hypotonic tubular fluid entering the early distal tubule (distal convoluted tubule) remains hypotonic to plasma until it enters the late distal tubule (initial collecting tubule) and that vasopressin-induced osmotic equilibration is a function of the latter segment alone. The findings emphasize the importance of morphological characterization of those segments of the renal tubule that are subjected to physiological investigation.

Relationship between para-aminohippurate secretion and cellular morphology in rabbit proximal tubules.

Previous studies in the mammalian proximal tubule have suggested that para-aminohippurate (PAH) secretion is approximately threefold greater in the straight segment, or pars recta, than in the convoluted segment, or pars convoluta. However, the possibility that the site of maximal PAH secretion might be related better to particular tubule segments as identified by cell type had not been explored. In addition, the presence or absence of differences in PAH secretion between morphologically identical regions of superficial (SF) vs. juxtamedullary (JM) proximal tubules has not been examined. These issues were studied using a combination of histologic methods and measurement of [(3)H]PAH secretion in isolated perfused tubules. Measurements of microdissected SF and JM proximal tubules from young and adult rabbits revealed that SF proximal tubules were slightly but significantly longer than JM tubules ([young rabbits: SF, 8.69+/-SE 0.14 mm vs. JM, 7.97+/-SE 0.13 mm; P < 0.01] [adult rabbits: SF, 10.61+/-SE 0.28 mm; JM, 9.17+/-SE 0.19 mm; P < 0.001]). Light and electron microscopy revealed three sequential segments (S(1), S(2), and S(3)) along the length of SF and JM proximal tubules as defined by cell type. PAH secretion was measured in each of these three segments by the isolated perfused tubule technique. Net PAH secretion in fmol/mm per min in SF proximal tubules was: S(1), 281+/-SE 21; S(2), 1,508+/-SE 104; S(3), 318+/-SE 46. Corresponding values in JM proximal tubules were 353+/-SE 31, 1,391+/-SE 72, and 188+/-SE 23. Net PAH secretion did not differ between comparable segments of SF and JM proximal tubules. It is concluded that differences in PAH secretion along the proximal tubule correlate best with cell type rather than the arbitrary division of the proximal tubule into pars convoluta and pars recta according to its external configuration. Evidence of functional heterogeneity between comparable segments of SF and JM proximal tubules was not observed.

Relationship between phosphaluria and acute hypercapnia in the rat.

Standard clearance studies were performed in mechanically ventilated intact and acutely thyroparathyroidectomized (TPTX) rats to document and characterize the effect of hypercapnia (HC) on urinary phosphorus excretion (U(P)V). HC as compared to normocapnia (NC) was associated with an increase in U(P)V in intact (62.5 vs. 7.93 mug/min) and TPTX (30.5 vs. 0.59 mug/min) rats, an increase in filtered load of phosphorus in intact (218 vs. 191 mug/min) and TPTX (243 vs. 146 mug/min) rats, an increase in blood bicarbonate concentration in intact (27.8 vs. 26.0 meq/liter) and TPTX (24.5 vs. 22.3 meq/liter) animals, and a decrease in blood pH in intact (7.15 vs. 7.42) and TPTX (7.07 vs. 7.39) rats. Additional TPTX rats with NC and HC were studied during phosphorus infusion at a comparable filtered load of phosphorus (NC = 307 mug/min and HC = 328 mug/min). U(P)V was 18.5 mug/min in NC and 85.2 mug/min in HC animals. Intact NC animals infused with NaHCO(3) achieved a blood bicarbonate of 45.9 meq/liter compared to 26.0 meq/liter in intact NC NaCl-infused rats. U(P)V was 10.0 mug/min in the NaHCO(3) and 7.93 mug/min in NaCl-infused animals. In intact HC animals infused with NaHCO(3), blood pH was 7.36 compared to 7.42 in NC intact NaCl-infused animals. U(P)V was 83.2 mug/min in the HC bicarbonate-infused and 7.93 mug/min in the NC NaCl-infused rats. These experiments demonstrate that elevated blood carbon dioxide tension per se increases U(P)V. Increases in filtered load of phosphorus and blood bicarbonate which are associated with HC contribute to the phosphaturia as does parathyroid hormone. The phosphaturia is not dependent upon reduction of extracellular pH.

Focal glomerular sclerosis: contrasting clinical patterns in children and adults.

In a retrospective clinicopathological study, 48 kidney biopsy specimens from 16 children (mean age, 7 years) and 17 adults (mean age, 33 years) with histological evidence of focal glomerular sclerosis (FGS) were examined using light, immunofluorescence and electron microscopy. The histopathological findings were related to the clinical course of each patient. At the clinical onset of the disease, the nephrotic syndrome was seen more commonly in children (12/16) than adults (7/17), while the incidence of both hypertension (children 1/16 versus adults, 9/17) and renal insufficiency (children, 0/16 versus adults, 7/17) was greater in adults. Despite a shorter average follow-up, (adults 3 10/12 years versus children, 7 years), the incidence of hypertension (adults, 13/17 versus children, 7/16) and renal functional impairment (adults, 13/17 versus children, 3/16) remained greater in the adult patients. One child and three adults died in renal failure while two adults underwent transplantation and on requires regular dialysis therapy. Nine of 15 pediatric patients treated with corticosteroids experienced partial or complete remission in either their nephrotic syndrome or level of urine protein excretion, while just 3 of 6 adult patients treated with corticosteroids experienced a partial remission, but never became protein-free. There was an excellent correlation in all patients between the degree of functional renal impairment and the extent of glomerular and nonglomerular histopathological damage in the kidney. It is concluded that in the adults, FGS represents a more severe and progressive disease process and is less responsive to therapy.

Renal transplantation between HL-A haploidentical donor-recipient pairs: functional and morphological evaluation.

Fifty-nine recipients received renal allografts from an HL-A haploidentical family member. Immunogenicity of the incompatible haplotype was measured by skin grafts exchanged within each family when possible, and renal allograft recipients were assigned prospectively to two groups depending on the skin graft survival time (Group 2A greater than 15 days; Group 2B less than 15 days). If skin grafts could not be accomplished, the patients were place in an unclassified group, Group 2. Renal function at one and 2 years following engraftment did not differ between the two groups. Mixed lymphocyte stimulation of recipient lymphocytes by mitomycin-treated donor lymphocytes also was comparable in the groups. Histopathological evaluation by light, immunofluorescence, and electron microscopy at least 6 months following allografting did not distinguish between the groups. The only differentiating characteristic was that Group 2A patients did not experience their primary rejection episode until an average of 18 days following transplantation, whereas Groups 2B and unclassified 2 had their initial primary rejection episode at average days 9 and 5, respectively. In our clinical program, matching for HL-A halotypes continues to be the best predictor for long-term renal function in consanguineous renal transplantation.

Axial distribution of band 3-positive intercalated cells in the collecting duct of control and ammonium chloride-loaded rabbits.

Recent studies have suggested that less than 10% of intercalated cells in the rabbit outer cortical collecting duct (CCD) [1, 2] and less than 3% in the connecting segment (CNT) [3] are identifiable by functional criteria as acid-secreting (type A or alpha) intercalated cells. Other studies, using peanut lectin-binding and the absence of apical endocytic activity to identify bicarbonate-secreting (type B or beta) intercalated cells, have suggested that acid-loading increases the percentage of alpha intercalated cells in the CCD. Because our preliminary observations of band 3 immunoreactivity suggest that the percentages of alpha intercalated cells in the rabbit outer CCD and the CNT are underestimated by physiologic studies and are not altered by chronic acid-loading, we quantified the percentage of alpha intercalated cells in various segments of the collecting duct using light microscopic immunohistochemistry in kidneys of rabbits receiving tap water (control) or 75 mM NH4Cl for 12 days plus 8 daily gavages of 2 to 6 mEq NH4Cl/kg body wt. Mean urine pH values were 5.96 in acid-loaded animals versus 8.47 in controls. Kidneys were preserved by in vivo perfusion with periodatelysine-paraformaldehyde fixation and processed for immunohistochemical colocalization using sequential labeling with monoclonal antibodies and peanut lectin, followed by immunoperoxidase detection. Colocalization of band 3 and carbonic anhydrase II immunoreactivity revealed the following percentages of band 3-positive intercalated cells in control versus NH4Cl rabbits: CNT, 49.0 versus 52.8; initial collecting tubule (ICT), 27.2 versus 34.5; outer CCD, 33.5 versus 30.3; inner CCD, 38.2 versus 41.8; corticomedullary CD, 67.9 versus 58.8. There were no differences between the groups for all comparisons. Similar results were obtained using band 3 protein immunoreactivity and peanut lectin-binding to identify intercalated cell subtypes. However, in NH4Cl-loaded rabbits, peanut lectin-binding was observed in band 3 positive intercalated cells in the outer medullary CD. We conclude that: (1) the percentage of alpha intercalated cells in rabbit CCD subsegments are approximately 50% in the CNT, 30% in the ICT and the outer CCD, 40% in the inner CCD, and 60% in the corticomedullary CD; (2) the percentage of alpha intercalated cells is not altered by chronic NH4Cl-loading; (3) peanut lectin is not a specific marker of beta intercalated cells.

Hemolytic uremic syndrome in dogs.

A disease syndrome similar to the hemolytic uremic syndrome of people is described in three dogs with acute renal failure. In each dog, hemorrhagic gastroenteritis preceded the onset of anuric acute renal failure. Evidence of microangiopathic hemolytic anemia (schizocytes, thrombocytopenia, and increased concentrations of fibrin split products) was present in the three dogs. Serum chemistry results showed increased concentrations of blood urea nitrogen, creatinine, and phosphorus. Ultrasound examination performed in one dog revealed increased echogenicity of the renal cortices. Treatment for anuric acute renal failure using a continuous dopamine and furosemide infusion established urine production in one of three dogs. Microscopic examination of tissue from the two dogs that underwent necropsy showed occlusion of the renal vasculature by fibrin thrombi consistent with microangiopathic arteriolar thrombosis. The pathophysiology and current knowledge of human hemolytic uremic syndrome is compared with hemolytic uremic syndrome in these dogs.

Adeno-associated viral vector-mediated interleukin-10 prolongs allograft survival in a rat kidney transplantation model.

Interleukin-10 (IL-10) is a pleiotropic cytokine that plays a pivotal role in the regulation of immune responses. Hence, we evaluated the effects of a recombinant adeno-associated viral vector 1 (rAAV1) encoding rat IL-10 (rAAV1-IL-10) in a rat model of kidney allograft rejection. Dark Agouti rat kidneys were transplanted into Wistar-Furth (WF) rats 8 weeks following a single intramuscular administration of either rAAV1-IL-10 or rAAV1-green fluorescence protein (GFP). Isografts (WF-WF) served as an additional experimental control. Both allograft and isograft recipients received daily cyclosporine (10 mg/kg) for 14 days after transplantation. Serum IL-10 levels increased at 8, 12 and 16 weeks following vector administration in rAAV1-IL-10-treated animals, but not in rAAV1-GFP and isograft groups. rAAV1-IL-10 treatment resulted in lower BUN and creatinine levels (p<0.001), as well as increased allograft survival rates from 22% to 90%. Allograft histological abnormalities were significantly attenuated in the rAAV1-IL-10-treated rats compared with those of rAAV1-GFP controls. Serum levels of proinflammatory cytokines such as growth-related oncogene were also significantly higher in the rAAV1-GFP group than in the rAAV1-IL-10 group. These data suggest delivery of IL-10 using a rAAV1 vector improves renal function and prolongs graft survival in a rat model of kidney transplant rejection.

Functional anatomy of the kidney.

Initiating a new series of articles on renal function in health and disease, this article describes the ultrastructure of the kidney's basic unit, the nephron, as revealed in recent years by transmission and scanning electron microscopy. With greater appreciation of its morphology and cellular architecture has come new understanding of the functional relationships among the nephron's individual components.

Response of intercalated cells of rat outer medullary collecting duct to chronic metabolic acidosis.

The outer medullary collecting duct which is composed of both principal and intercalated cells is involved in hydrogen ion secretion. In the turtle urinary bladder stimulation of hydrogen ion secretion is associated with ultrastructural changes in the mitochondria-rich cells, suggesting that membrane and possibly a proton pump are being transferred from apical tubulovesicular structures and inserted into the apical plasma membrane. Since the intercalated cells resemble the mitochondria-rich cells, this study was initiated to determine whether or not similar changes occur in the outer medullary collecting duct during chronic metabolic acidosis. Rats received ammonium chloride in their drinking water for 15 days and as a daily gavage for 3 days before sacrifice. Control rats received regular tap water. After collection of physiologic data the kidneys were fixed by in vivo perfusion with glutaraldehyde and processed for electron microscopy. No changes were observed in the principal cells. Morphometric analyses of the intercalated cells in both the outer and inner stripe revealed a significant increase in the surface density of the apical plasma membrane concomitant with a striking depletion of the tubulovesicular structures in the apical plasma region of the cell with chronic metabolic acidosis. These findings suggest that in response to chronic metabolic acidosis membrane, possibly containing a proton pump, is transported from the tubulovesicular membrane compartment to the apical plasma membrane of the intercalated cell.

Structure-function relationships in H+-secreting epithelia.

Stimulation or inhibition of H+ secretion has been associated with characteristic ultrastructural changes in various epithelial cells, including the parietal cell of the gastric mucosa, the carbonic anhydrase (CA)-rich cell of the turtle urinary bladder, and the intercalated (I) cell of the mammalian collecting duct. An electroneutral potassium-activated H+-ATPase is responsible for H+ secretion in the stomach, whereas acidification in the turtle bladder and the mammalian collecting duct is mediated by an electrogenic H+-translocating ATPase. Despite these differences, the parietal cell, the CA-rich cell, and the I cell have several morphological features in common. They are rich in mitochondria, contain numerous tubulovesicular membrane structures in the apical region of the cell, and possess a variable number of microprojections on the luminal surface. After stimulation of H+ secretion there is a significant increase in the surface area of the apical membrane concomitant with a decrease in the tubulovesicular membrane compartment in these cells, as revealed by morphometric analysis. These findings suggest that membrane (possibly containing an H+ pump) is being transferred from the tubulovesicular compartment to the apical plasma membrane on stimulation of H+ secretion. A hypothesis of membrane recycling is proposed to account for the observed morphological changes.

Morphology of rabbit collecting duct.

Recently the assumed structural and functional homogeneity of the collecting duct (CD) has been questioned. The objective of this study was to determine if heterogeneity occurs in luminal surface membrane structure or in cytoplasmic configuration of cells in the collecting duct or both. Straight segments of cortical and medullary CD were examined in perfusion-fixed rabbit kidneys with scanning electron microscopy (SEM), light (LM) and transmission electron microscopy (TEM). Principal cells were the most abundant cells in all CD regions; intercalated cells comprised 37% of the cell population on the cortex, 18% in the outer medulla, and less than 1% in the inner medulla. SEM revealed two surface patterns among the ciliated principal cells: 1, located in the cortex and outer medulla, with few surface microvilli, and 2, located in the inner medulla, with abundant microvilli. Intercalated cells exhibited four distinctive luminal surface configurations: I, numerous short microvilli; II, both short and elongate microvilli; III, microplicae alone; and IV, both microvilli and microplicae. Intercalated cells with patterns I and II were predominant in the cortex, while cells with patterns III and IV were most common at the corticomedullary junction. TEM confirmed that marked variation existed in cytoplasmic structures of both principal and intercalated cells. These findings may either indicate the presence of several specific types of principal and intercalated cells or reflect different functional states of the principal and intercalated cells. Regardless of their significance, their presence must be considered in studies seeking to establish precise structural-functional relationships in this region of the rabbit renal tubule.