Daniel R. Martin

Combination Therapy with an Angiotensin-Converting Enzyme Inhibitor and a Vitamin D Analog Suppresses the Progression of Renal Insufficiency in Uremic Rats

Monotherapy with angiotensin-converting enzyme inhibitors has been shown to be beneficial in suppressing the progression of experimentally induced kidney diseases. Whether such therapy provides additional benefits when combined with vitamin D or an analog of vitamin D has not been established. Rats were made uremic by 5/6 nephrectomy and treated as follows: Uremic + vehicle (UC), uremic + enalapril (30 mg/L in drinking water; E), uremic + paricalcitol (19-nor; 0.8 microg/kg, three times a week), and uremic + enalapril + paricalcitol (E + 19-nor). A group of normal rats served as control (NC). BP was significantly elevated in the UC and 19-nor groups compared with the NC group but was indistinguishable from normal in the E and E + 19-nor groups. The decrease in creatinine clearance and the increase in the excretion of urinary protein that were observed in the UC group were ameliorated by the use of E alone or by E + 19-nor (P < 0.05 versus UC). The glomerulosclerotic index was significantly decreased in both the 19-nor (P < 0.01) and E + 19-nor groups (P < 0.01) compared with the UC group. Tubulointerstitial volume was significantly decreased in both the E (P < 0.05) and E + 19-nor groups (P < 0.01) compared with the UC group. Both macrophage infiltration (ED-1-positive cells) and production of the chemokine monocyte chemoattractant protein-1 were significantly blunted in E + 19-nor compared with E group. TGF-beta1 mRNA and protein expression were increased in the UC group (mRNA: 23.7-fold; protein: 29.1-fold versus NC). These increases were significantly blunted in the 19-nor group (mRNA: 7.1-fold; protein: 8.0-fold versus NC) and virtually normalized in the E + 19-nor group (protein: 0.8-fold versus NC). Phosphorylation of Smad2 was also elevated in the UC group (7.6-fold versus NC) but less so in the 19-nor-treated rats (5.5-fold versus NC). When rats were treated with E + 19-nor, the phosphorylation of Smad2 was normal (1.1-fold versus NC). Thus, 19-nor can suppress the progression of renal insufficiency via mediation of the TGF-beta signaling pathway, and this effect is amplified when BP is controlled via renin-angiotensin system blockade.

Cloning of wrinkle-free, a previously uncharacterized mouse mutation, reveals crucial roles for fatty acid transport protein 4 in skin and hair development

Wrinkle-free (wrfr) is a previously uncharacterized, spontaneous, autosomal recessive mouse mutation resulting in very tight, thick skin. wrfr mutant mice exhibit severe breathing difficulties secondary to their tight skin and die shortly after birth. This phenotype is strikingly similar to a very rare human genetic disorder, restrictive dermopathy. wrfr mutant mice display a defective skin barrier, which is normally imparted by the cornified envelope, a composite of protein and lipid that prevents loss of water from within and entry of potentially harmful substances from without. In addition, hair growth from grafted wrfr skin is impaired. Positional cloning of the wrfr mutation revealed a retrotransposon insertion into a coding exon of Slc27a4, the gene encoding fatty acid transport protein (FATP)4. FATP4 is the primary intestinal FATP and is thought to play a major role in dietary fatty acid uptake; it therefore is viewed as a target to prevent or reverse obesity. However, its function in vivo had not been determined. Our results demonstrate an unexpected yet critical role for FATP4 in skin and hair development and suggest Slc27a4 to be a candidate gene for restrictive dermopathy.

Activation of NFAT Signaling in Podocytes Causes Glomerulosclerosis

Mutant forms of TRPC6 can activate NFAT-dependent transcription in vitro via calcium influx and activation of calcineurin. The same TRPC6 mutants can cause FSGS, but whether this involves an NFAT-dependent mechanism is unknown. Here, we generated mice that allow conditional induction of NFATc1. Mice with NFAT activation in nascent podocytes in utero developed proteinuria and glomerulosclerosis postnatally, resembling FSGS. NFAT activation in adult mice also caused progressive proteinuria and FSGS. Ultrastructural studies revealed podocyte foot process effacement and deposition of extracellular matrix. NFAT activation did not initially affect expression of podocin, synaptopodin, and nephrin but reduced their expression as glomerular injury progressed. In contrast, we observed upregulation of Wnt6 and Fzd9 in the mutant glomeruli before the onset of significant proteinuria, suggesting a potential role for Wnt signaling in the pathogenesis of NFAT-induced podocyte injury and FSGS. These results provide in vivo evidence for the involvement of NFAT signaling in podocytes, proteinuria, and glomerulosclerosis. Furthermore, this study suggests that NFAT activation may be a key intermediate step in the pathogenesis of mutant TRPC6-mediated FSGS and that suppression of NFAT activity may contribute to the antiproteinuric effects of calcineurin inhibitors.

Extracellular matrix-related genes in kidney after ischemic injury : potential role for TGF-β in repair

The renal expression of transforming growth factor-β1 (TGF-β1) is enhanced following induction of ischemic injury in rat. In cultured renal cells, TGF-β stimulates the synthesis of extracellular matrix. To link TGF-β1 expression with the regulation of extracellular matrix postischemia, we characterized the expression of several genes known to regulate extracellular matrix synthesis at various times during recovery from acute ischemic renal injury in rat. Levels of mRNA for plasminogen activator inhibitor-1 (PAI-1), tissue inhibitor of metalloprotease-1 (TIMP-1), α1(IV) collagen, and fibronectin-EIIIA (FN-EIIIA) mRNAs were significantly enhanced in kidneys within 12 h to 3 days after injury and remained elevated at 7-28 days postischemia relative to levels in kidneys of sham-operated controls. PAI-1 mRNA and peptide were localized in regenerating proximal tubules at 3 and 7 days postischemic injury. α1(IV) Collagen and FN-EIIIA mRNAs were expressed primarily in regenerating proximal tubule cells. Immunoreactivity for FN-EIIIA was enhanced in the tubular basement membrane (TBM) of regenerating proximal tubules, and α1(IV) collagen immunoreactivity was detected in thickened tubulointerstitial spaces. In contrast, TIMP-1 immunoreactivity was enhanced in distal nephron structures postischemia. Immunoneutralization of TGF-β in vivo attenuated the increases in FN-EIIIA, α1(IV) collagen, PAI-1, and TIMP-1 mRNAs by 52%, 73%, 43%, and 27%, respectively. These data are consistent with TGF-β expression postischemic injury participating in renal regeneration of extracellular matrix homeostasis in the proximal TBM.

Reversal of Secondary Hyperparathyroidism by Phosphate Restriction Restores Parathyroid Calcium-Sensing Receptor Expression and Function

Secondary hyperparathyroidism (2° HPT), a common disorder in chronic renal failure (CRF) patients, is characterized by hypersecretion of parathyroid hormone (PTH), parathyroid hyperplasia, and decreased expression of the calcium‐sensing receptor (CaR). Dietary phosphate loading promotes 2° HPT, and phosphate restriction prevents and arrests 2° HPT in CRF. This study examined the ability of phosphate restriction to restore parathyroid CaR expression and function. Uremic rats fed a 1.2% P diet for 2 weeks developed 2° HPT with down‐regulated CaR expression. Continuation on the 1.2% P diet for 2 more weeks worsened the 2° HPT and further decreased CaR, but switching the rats to a 0.2% P diet for 2 weeks normalized PTH, arrested parathyroid hyperplasia, and restored CaR expression to normal. The calcium‐PTH relationship was abnormal in uremic rats fed a high phosphate (HP) diet with a right‐shifted calcium set point but was corrected by 2 weeks of phosphate restriction. A time course revealed that following the switch to a low phosphate diet, PTH levels were normalized by day 1, and growth was arrested by day 2, but CaR expression was restored between days 7 and 14. We conclude that although phosphate restriction restores CaR expression and function in parathyroid glands of uremic rats, it is a late event and not involved in the arrest of 2° HPT.

Whole body leptin kinetics and renal metabolism in vivo

Leptin metabolism was investigated in male Sprague-Dawley rats by use of125I-labeled leptin plasma kinetic and arteriovenous balance studies. When conscious rats received bolus venous injections of 125I-leptin, intact (precipitable) leptin quickly disappeared from circulation in a biexponential manner during the 2-h experimental period. After substantial delay, most of the injected radioactivity appeared in the urine. The data were described by a two-compartment model, which postulated that plasma leptin exchanged with a nonplasma pool and that all of the tracer cleared from plasma appeared in urine or in a degraded form in plasma. The half-life of leptin was 9.4 ± 3.0 min, and the leptin production rate was 3.6 ± 1.2 ng ⋅ 100 g fat-1 ⋅ min-1. The left kidney extracted 21 ± 1.5% of intact arterial125I-leptin 5 min after femoral venous injection. Endogenous arterial leptin was reduced 21 ± 8 and 18 ± 12%, respectively, in simultaneously sampled left and right renal veins. Renal elimination appears to be the major elimination mechanism for leptin in normal rats, and the kinetic studies suggest that uptake of leptin by renal tissue rather than glomerular filtration is the predominant elimination mechanism.

Ammonium Handling by Superficial and Juxtamedullary Nephrons in the Rat: EVIDENCE FOR AN AMMONIA SHUNT BETWEEN THE LOOP OF HENLE AND THE COLLECTING DUCT

Papillary and surface micropuncture was used to assess the effects of a chronic metabolic acidosis on the renal tubular handling of ammonium by surface nephrons, juxtamedullary nephrons, and the terminal segment of collecting duct. Rats chronically fed ammonium chloride had an expected decline in arterial pH and bicarbonate concentration associated with a doubling in the amount of ammonium excreted and a decline in urine pH. The glomerular filtration rate and absolute delivery of water and sodium to micropuncture sites of surface and deep nephrons was not measurably altered. Ammonium delivery to the end of the proximal tubule increased from 853+/-102% to 1,197+/-142% (SE) of the filtered load of ammonium after the induction of metabolic acidosis. This increase was due to a rise in tubular fluid ammonium content from 2.31+/-0.23 to 4.06+/-0.28 mM/liter. After the induction of acidosis, absolute and fractional delivery of ammonium ion to the end of the distal tubule was less than to the end of the accessible portion of the proximal tubule. These findings indicate that ammonium is lost in the intervening segment.Ammonium handling by deep nephrons was profoundly affected by acid loading. Absolute delivery to the bend of the loop of Henle increased twofold while fractional delivery rose from 1,222+/-108% to 1,780+/-132% of the filtered ammonium. This was due to a marked increase in ammonia entry. During acidosis, ammonium delivery to the terminal segment of the collecting duct was doubled (709+/-137% in controls vs. 1,415+/-150% in acidosis, P < 0.005) but did not change between proximal and tip collecting duct sites. In both groups of animals delivery of ammonium to the terminal segment of the collecting duct was greater than to end distal tubular micropuncture sites suggesting that ammonia entry occurred between these two sites. The differences in delivery was greater after the induction of a metabolic acidosis (887+/-140% vs. 384+/-144%, P < 0.05). Thus, the present study indicates that deep nephrons contribute to the adaptive increase in ammonium excretion seen during the induction of metabolic acidosis. The data also suggest that ammonia leaves the nephrons at a site(s) along the loop of Henle to enter the collecting duct and that the induction of a metabolic acidosis enhances this reentry.

Effect of Reduced Renal Mass on Ammonium Handling and Net Acid Formation by the Superficial and Juxtamedullary Nephron of the Rat: EVIDENCE FOR IMPAIRED REENTRAPMENT RATHER THAN DECREASED PRODUCTION OF AMMONIUM IN THE ACIDOSIS OF UREMIA

Papillary and surface micropuncture were used to study the handling of ammonium and the formation of net acid by surface nephrons, deep nephrons, and the terminal segment of collecting duct (CD) after renal mass was reduced by two-thirds. Net acid excretion by the remnant kidney (RK) was significantly reduced, averaging 794+/-81 neq/min (SE) compared with 1,220+/-105 neq/min after sham operation (P < 0.001), due to a decrease in ammonium excretion (494+/-54 vs. 871+/-79 nmol/min in controls, P < 0.001). Urinary pH and titratable acid excretion were not different in the two groups of animals. After RK formation, ammonium delivery to the end of the proximal tubule increased nearly threefold and averaged 66.2+/-5.6 compared with 18.4+/-2.9 pmol/min in controls, (P < 0.001). This greater delivery of ammonium was primarily due to renal tubule entry rather than to changes in the filtered load and was only partially related to the differences in flow rate. Ammonium processing by deep nephrons was profoundly affected by a reduction in renal mass. Although absolute delivery of ammonium was greater to the bend of Henles loop (BHL), the difference could be accounted for on the basis of an increase in nephron size. Thus, fractional delivery (FD(NH+4)) to this site was not different for the two groups of animals, averaging 1,567+/-180% in controls and 1,400+/-181% in the group with the RK. Hydrogen secretion in the proximal segments of deep and surface nephrons did not increase in proportion to the decrease in renal mass and as a consequence bicarbonate delivery to the end of the proximal tubule of surface nephrons and to the BHL of deep nephrons was increased. When renal mass was reduced FD(NH+4) to the base of the terminal CD doubled but did not change by the tip. In both groups FD(NH+4) to the base of the CD was greater than to the end of the distal tubule. However, the increase was the same. On the other hand, the increase in the net acid index between the end of the distal tubule and the base of the CD was profoundly greater in rats with an RK. This difference was primarily due to bicarbonate reabsorption rather than enhanced ammonium reentry. Indeed, >400% of the fractional ammonium delivered to the end of the proximal tubule was lost from the tubule fluid. The data suggest that the decrease in acid excretion by the RK is due to two factors. First, hydrogen secretion in the proximal segments of both nephron populations fails to increase in the proportion to the reduction in renal mass. Second, a reduced reentrapment of ammonia, rather than its impaired production, causes ammonium excretion to decrease.

Deep nephron function after release of acute unilateral ureteral obstruction in the young rat.

The effects of acute unilateral ureteral obstruction (UUO) of 18 h duration on deep nephron function was evaluated in 14 weanling rats with the technique of micropuncture. After release of UUO, 3.4 +/- 0.66% (SE) of the filtered water remained at the tip of the collecting duct nearly fivefold greater than in controls (0.75 +/- 0.10%). Similar differences were seen in fractional sodium that remained at this site. The ratio of tubular fluid osmolality to that of plasma was also reduced in the UUO group (1.53 +/- 0.06 vs. 4.60 +/- 0.26 in controls, P less than 0.001). Single nephron glomerular filtration rate of cortical and deep nephrons was significantly less (P less than 0.001) after release of UUO. Although the percentage of filtering nephrons was significantly reduced in both nephron populations, the decline in glomerular filtration rate was greater in cortical than in juxtamedullary nephrons (cortical:juxtamedullary nephrons = 27.6 +/- 4.5% vs. 53.3 +/- 5.2% in controls, P less than 0.005) which suggests that single nephron glomerular filtration rate is redistributed to deep nephrons after release of UUO. In contrast to cortical nephrons, the amount of tubular fluid which remains near the bend of the loop of Henle of deep nephrons was greater after release of UUO. This appeared to be the result of a decrease in the reabsorption of both water (tubular fluid:plasma inulin = 2.41 +/- 0.16 vs. 7.94 +/- 0.69 in controls, P less than 0.001) and sodium (52.3 +/- 4% vs. 40.7 +/- 2.9% of the filtered sodium in controls, P less than 0.02). It is suggested that this altered reabsorption occurs along both the proximal tubule and descending limb of the loop of Henle of juxtamedullary nephrons. Inner medullary plasma flow (IMPF), as measured with the [125I]albumin-accumulation technique, was significantly depressed before release of UUO, but exceeded control values 90 min postrelease. Such changes imply that the filtration fraction of deep nephrons is decreased and that physical factors in the proximal tubular reabsorption of sodium have been altered. When papillary solute content was measured before release of UUO it was low (428 +/- 23 vs. 1,205 +/- 106 mosmol/kg in controls, P less than 0.001) which indicates that the decline in papillary osmolality is not a consequence of the increased IMPF seen after ureteral release, but rather precedes it. In fact, the decline in papillary osmolality may contribute to the increase in IMPF after release of UUO and to the decreased reabsorption of fluid along the descending limb of the loop of Henle.

Transport of ammonia in the rabbit cortical collecting tubule.

Nonionic diffusion and diffusion equilibrium of ammonia have been generally accepted as the mechanism of urinary ammonium excretion. However, these characteristics have not been examined directly in vitro. In the present studies, nonionic diffusion and diffusion equilibrium of ammonia were examined in rabbit cortical collecting tubules perfused in vitro. Collected fluid ammonium and pH were measured in tubules exposed to chemical gradients of NH3/NH+4. In tubules perfused with an acid perfusate free of ammonia and bathed with solutions containing NH4Cl, collected fluid ammonia failed to equilibrate across the epithelium except at slow flow rates. The estimated apparent permeability coefficient to NH3 was approximately 5 X 10(-3) cm/s. Predominant nonionic diffusion of NH3, rather than transport of NH+4, was indicated by alkalinization of luminal fluid in tubules exposed to peritubular NH4Cl and by the relative influence of peritubular NH+4 and NH3 on ammonia entry. In tubules perfused with an acid solution containing NH4Cl, little loss of ammonium was detectable, indicating a low permeability to NH+4. In contrast to the restricted diffusion of NH3 in cortical collecting tubules, proximal convoluted tubules exhibited a much higher apparent permeability to NH3. In conclusion, nonionic diffusion of NH3 accounted for most ammonium transport in the proximal convoluted tubule and in the cortical collecting tubule. However, there was relatively restricted diffusion in the collecting tubules; this may account for the failure of whole kidney ammonium excretion to obey quantitatively the predictions of nonionic diffusion and diffusion equilibrium of ammonia.