C. Craig Tisher

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.

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.

Recurrent Diseases in the Kidney Transplant

Virtually all diseases affecting the native kidney recur in the kidney transplant with the exception of Alport syndrome, polycystic kidney disease, hypertension, chronic pyelonephritis, and chronic interstitial nephritis. Fortunately, in the majority of patients, recurrence of the original disease has minimal clinical impact, with only approximately 5% of all graft loss occurring as a result of recurrent disease. The primary renal diseases that commonly recur include membranoproliferative glomerulonephritis type II, IgA nephropathy, and focal and segmental glomerular sclerosis. The most common systemic disease that recurs is diabetic nephropathy. Living-related transplantation should be used with caution in patients with the hemolytic uremic syndrome, recurrent focal and segmental glomerular sclerosis, and membraneous glomerulonephritis. Fabry disease and primary hyperoxaluria type I are no longer absolute contraindications to kidney transplantation.

Uncoupling of Vascular Endothelial Growth Factor with Nitric Oxide as a Mechanism for Diabetic Vasculopathy

The role of VEGF in vascular disease is complicated. Vascular endothelial growth factor (VEGF) expression can be deleterious in diabetic vasculopathy, especially in kidney and retina. In contrast, VEGF seems to be renoprotective in nondiabetic renal disease. VEGF exerts it biologic effects in association with nitric oxide (NO), yet it is known that NO bioavailability is reduced in diabetes. Thus, it was hypothesized that this diverse biologic effect of VEGF on diabetic vasculopathy is due to uncoupling of VEGF with NO. VEGF stimulated NO production in a dose-dependent manner in bovine aortic endothelial cells (BAEC), and this was inhibited by either high glucose or Nomega-nitro-l-arginine methyl ester (L-NAME) treatment. Endothelial NO synthase phosphorylation by VEGF was also inhibited by high glucose. It is interesting that both high glucose and L-NAME enhanced the proliferative response of endothelial cells, which was prevented by an NO donor. Furthermore, high glucose as well as L-NAME stimulated VEGF and kinase-insert domain receptor (KDR) (VEGF receptor 2) mRNA expression in BAEC. These data suggest that the uncoupling of VEGF with NO enhances endothelial cell proliferation via the KDR pathway. Compatible with these findings, a KDR antagonist blocked this response. In addition, a VEGF mutant, which binds only KDR, induced extracellular signal-regulated kinase (ERK) activation, and inhibition of ERK completely blocked endothelial cell proliferation under this condition, suggesting a role of the KDR-ERK1/2 pathway on endothelial cell proliferation. In conclusion, high glucose causes an uncoupling of VEGF with NO, which enhances endothelial cell proliferation via activation of the KDR-ERK1/2 pathway. These results may provide new insights into the understanding of the mechanism of diabetic vascular disease.

Gene Delivery in Renal Tubular Epithelial Cells Using Recombinant Adeno-Associated Viral Vectors

Gene therapy has the potential to provide a therapeutic strategy for numerous renal diseases such as diabetic nephropathy, chronic rejection, Alport syndrome, polycystic kidney disease, and inherited tubular disorders. In previous studies using cationic liposomes or adenoviral or retroviral vectors to deliver genes into the kidney, transgene expression has been transient and often associated with adverse host immune responses, particularly with the use of adenoviral vectors. The unique properties of recombinant adeno-associated viral (rAAV) vectors permit long-term stable transgene expression with a relatively low host immune response. The purpose of the present study was to evaluate gene expression in the rat kidney after intrarenal arterial infusion of a rAAV (serotype 2) vector encoding green fluorescence protein (GFP) induced by a cytomegalovirus-chicken beta-actin hybrid promoter. The left kidney of experimental animals was treated with either saline or transduced with rAAV2-GFP (0.125 ml/100 g body wt, 1 x 10(10)/ml infectious units) through the renal artery. A time-dependent expression of GFP was observed in all kidneys injected with rAAV2-GFP, with maximal expression observed at 6 wk posttransduction. The expression of GFP was restricted to cells in the S(3) segment of the proximal tubule and intercalated cells in the collecting duct, the latter identified by co-localization with H(+)-ATPase. No transduction was observed in the glomeruli or the intrarenal vasculature. These studies demonstrate successful transgene expression in tubular epithelial cells, specifically in the S(3) segment of the proximal tubule and intercalated cells, after intrarenal administration of a rAAV vector and provide the impetus for further studies to exploit its use as a tool for gene therapy in the kidney.

Effect of Low Potassium-Diet on Na-K-ATPase in Rat Nephron Segments

Na−K-ATPase activity was determined in 10 segments of the rat nephron using a fluorometric microassay method [4]. The enzyme activity showed three peaks (>200 pmol ADP min−1 mm−1) along the nephron of normal rats. These peaks were in the S1 portion of the proximal tubule, the medullary thick ascending limb from the inner stripe and the distal convoluted tubule. Feeding the rats a low potassium diet for 8 weeks produced a significant decrease in Na−K-ATPase activity in the cortical collecting duct, but no significant change in this enzyme in any other segment. The low potassium diet did not produce a significant change in Mg-ATPase in any nephron segments. We conclude that Na−K-ATPase activity along the rat nephron shows a pattern that is qualitatively similar to that seen in the rabbit nephron [4]. However, quantitatively the Na−K-ATPase activity in the rat nephron is greater than in the corresponding segments of the rabbit nephron. The results are consistent with the greater rate of glomerular filtration and Na+ reabsorption per rat nephron. Furthermore, our results suggest that the decrease in potassium excretion during potassium deficiency is modulated, at leat in part, by the level of Na−K-ATPase activity in the cortical collecting duct.

Intraendothelial Inclusions in Kidneys of Patients with Systemic Lupus Erythematosus

Abstract Recent renal biopsy material was reviewed to determine the relationship between intraendothelial virus-like structures and a diagnosis of lupus erythematosus, to establish the specificity ...

Relationship between Phosphaturia 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.

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.

Phagocytosis of erythrocytes by the proximal tubule of the rat kidney

SummaryMorphological examination of kidney biopsies from patients with glomerulonephritis and hematuria has revealed the presence of erythrocytes within epithelial cells of the proximal tubule. This observation suggested that the proximal tubule might be capable of phagocytizing morphologically intact erythrocytes. To examine this possibility small quantities of heparinized autologous blood were injected into surface convolutions of proximal tubules of the rat kidney using standard micropuncture techniques. At time intervals ranging from 10 min to 120 h after injection, the kidneys were preserved for light and transmission electron microscopy by drip-fixation with a half-strength Karnovskys glutaraldehyde-formaldehyde fixative.During the initial 6 h there was a flattening of the brush border and accumulation of electron-dense material representing hemoglobin in apical vacuoles and in lysosome-like structures. From 6 to 15 h after micropuncture, there was progressive loss of the brush border and the simultaneous formation of pseudopodia-like evaginations that extended from the apical plasma membrane and surrounded the individual erythrocytes. By 18 and 24 h, erythrocytes were observed in the proximal tubule cells. At later time intervals, edema, lymphocytic infiltration, and fibrosis were observed in the interstitium. In addition, crystalline structures were present in the lumen and the cells of both proximal and distal tubules. These findings suggest that in addition to their well-established ability to pinocytize hemoglobin and other proteins, the cells of the proximal tubule are capable of phagocytizing morphologically intact autologous erythrocytes. It is possible that phagocytosis by the proximal tubule cells may play a role in the disposal of erythrocytes from the tubular fluid in hematuric conditions.