Harry R. Jacobson

Effect of restricting dietary protein on the progression of renal failure in patients with insulin-dependent diabetes mellitus

BACKGROUND Restriction of dietary protein may slow the progression of renal failure in diverse renal diseases, but the extent to which such a diet is beneficial in patients with diabetic nephropathy is uncertain. METHODS We studied the effect of reduced intake of protein and phosphorus on the progression of renal disease in 35 patients with insulin-dependent (Type I) diabetes mellitus and clinically evident nephropathy. The low-protein, low-phosphorus diet contained 0.6 g of protein per kilogram of ideal body weight per day, 500 to 1000 mg of phosphorus, and 2000 mg of sodium. The control diet consisted of the patients prestudy diet with the stipulation that it contain 2000 mg of sodium and at least 1 g of protein per kilogram per day and 1000 mg of phosphorus. Renal function was assessed by measurement of iothalamate and creatinine clearances at intervals of 3 to 6 months, and the patients were followed for a minimum of 12 months (mean, 34.7). The declines in mean glomerular filtration rates were compared between groups by linear-regression analysis of the glomerular filtration rate as a function of time. RESULTS The patients who followed the study diet for a mean of 37.1 months had declines in iothalamate clearance of 0.0043 ml per second per month and in creatinine clearance of 0.0055 ml per second per month. The comparable values in the control group were 0.0168 and 0.0135, respectively (P less than 0.05). Blood pressure was well controlled, and the degree of glycemic control was comparable in both groups. CONCLUSION Dietary restriction of protein and phosphorus can retard the progression of renal failure in patients with Type I diabetes mellitus who have nephropathy. We believe that wider use of this treatment is indicated.

Angiotensin II directly stimulates sodium transport in rabbit proximal convoluted tubules.

Numerous previous studies have proposed a role for angiotensin II (AII) in the renal regulation of salt balance. At least one nephron site, the proximal convoluted segment, has been implicated in this role. We used in vitro microperfusion of rabbit proximal convoluted tubules to further examine this question. To insure use of appropriate in vivo concentrations as well as potency of the hormone in vitro, we measured plasma AII levels by radioimmunoassay in normal, sodium-depleted, and adrenalectomized rabbits, and measured AII activity by bioassay after incubation in various microperfusion baths. Plasma levels ranged from approximately 2 X 10(-11) to 5 X 10(-11) M. AII activity was stable in Ringers solution plus albumin, but not in rabbit serum or Ringers solution plus fetal calf serum. In Ringers solution plus albumin, physiologic concentrations of AII stimulated volume reabsorption (Jv). 10(-11) M AII increased Jv by 16% (P less than 0.01). 10(-10) M AII produced a lesser increase, 7.5% (P less than 0.05). At a frequently studied, but probably pharmacologic dose, 10(-7) M AII inhibited Jv by 24% (P less than 0.001). AII at 10(-11) M did not stimulate Jv in the presence of 10(-7) M saralasin. Though previous studies have suggested agonistic effects of saralasin alone in epithelia, we found no significant effect of 10(-7) M saralasin on Jv. None of the AII doses measurably changed transepithelial voltage. We conclude that AII in physiologic doses directly stimulates Jv in proximal convoluted tubules and this effect is probably receptor mediated and, within the limits of detection, electroneutral.

Experimental and/or genetically controlled alterations of the renal microsomal cytochrome P450 epoxygenase induce hypertension in rats fed a high salt diet.

Excess dietary salt induces a cytochrome P450 arachidonic acid epoxygenase isoform in rat kidneys (Capdevila, J. H., S. Wei, J. Yang, A. Karara, H. R. Jacobson, J. R. Falck, F. P. Guengerich, and R. N. Dubois. 1992. J. Biol. Chem. 267:21720-21726). Treatment of rats on a high salt diet with the epoxygenase inhibitor, clotrimazole, produces significant increases in mean arterial blood pressure (122 +/- 2 and 145 +/- 4 mmHg for salt and salt- and clotrimazole-treated rats, respectively). The salt- and clotrimazole-dependent hypertension is accompanied by reductions in the urinary excretion of epoxygenase metabolites and by a selective inhibition of the renal microsomal epoxygenase reaction. The prohypertensive effects of clotrimazole are readily reversed when either the salt or clotrimazole treatment is discontinued. The indication that a salt-inducible renal epoxygenase protects against hypertension, are supported by studies with the Dahl rat model of genetic salt-sensitive hypertension. Dahl resistant animals responded to excess dietary salt by inducing the activity of their kidney microsomal epoxygenase(s) (0.102 +/- 0.01 and 0.240 +/- 0.04 nmol of products formed/min per mg of microsomal protein for control and salt-treated rats, respectively). Despite severe hypertension during excess dietary salt intake (200 +/- 20 mmHg), Dahl salt-sensitive rats demonstrated no increase in renal epoxygenase activity. These studies indicate that acquired or inherited abnormalities in renal epoxygenase activities and/or regulation can be related to salt-sensitive hypertension in rodents. Studies on the human renal epoxygenase and its relationship to salt hypertension may prove useful.

Mineralocorticoid modulation of rabbit medullary collecting duct acidification. A sodium-independent effect.

Rabbit medullary collecting duct (MCD) from inner stripe of outer medulla has been identified as a major distal nephron acidification site. The isolated, perfused tubule technique was used to examine the roles of mineralocorticoid and glucocorticoid in regulation of MCD acidification. Surgical adrenalectomy reduced bicarbonate reabsorptive rate (JHCO3, pmol X mm-1 X min-1) from the normal of 9.79 +/- 1.21 to 0.67 +/- 1.1. Chronic administration of deoxycorticosterone acetate (DOCA) increased JHCO3 of MCD significantly to 18.02 +/- 1.62 whereas chronic dexamethasone administration did not affect JHCO3. The direct effects of aldosterone and dexamethasone upon MCD acidification were examined by perfusing tubules harvested from adrenalectomized rabbits in the presence of aldosterone or dexamethasone. Aldosterone, at 5 X 10(-8) M, increased JHCO3 significantly from 1.27 +/- 0.28 to 3.09 +/- 0.34. At 10(-6) M, aldosterone produced a greater increase in JHCO3 from 0.67 +/- 1.1 to 9.39 +/- 1.59. In vitro dexamethasone treatment had no effect on JHCO3. Studies examining the sodium dependence of aldosterone-stimulated acidification demonstrated that JHCO3 in tubules harvested from normal and deoxycorticosterone acetate-treated animals was unaffected by total replacement of sodium with tetramethylammonium. Likewise, luminal amiloride (5 X 10(-5) M) had no effect on JHCO3 in tubules harvested from adrenalectomized and normal animals. Moreover, the acute, in vitro stimulatory effect of aldosterone was seen to occur in the presence of luminal amiloride. These studies define a mammalian distal nephron segment that possesses major acidifying capacity, which is modulated by mineralocorticoid but independent of luminal sodium.

Prostaglandin E2 inhibits sodium transport in rabbit cortical collecting duct by increasing intracellular calcium.

The mechanism by which prostaglandin E2 (PGE2) inhibits sodium absorption (JNa) in the rabbit cortical collecting duct (CCD) was explored. PGE2 activates at least three signaling mechanisms in the CCD: (a) by itself PGE2 increases cAMP generation (b) PGE2 also inhibits vasopressin-stimulated cAMP accumulation, and (c) PGE2 raises intracellular calcium([Ca++]i). We tested the contribution of these signaling pathways to PGE2s effect on Na+ absorption, measuring 22Na flux (JNa) and [Ca++]i (using fura-2) in microperfused rabbit CCDs. In control studies PGE2 reduced JNa from 28.2 +/- 3.4 to 15.6 +/- 2.6 pmol.mm-1.min-1. Lowering bath calcium from 2.4 to 45 nM did not by itself alter JNa but in this setting PGE2 failed to inhibit JNa (28.6 +/- 5.4 to 38.5 +/- 4.0). In separate tubules, PGE2 raised [Ca++]i in a spike-like fashion followed by a sustained elevation. However, in 45 nM bath Ca++, PGE2 failed to produce a sustained [Ca++]i elevation. While pretreatment of CCDs with pertussis toxin blocked PGE2 inhibition of vasopressin-stimulated water permeability, it did not block the effect of PGE2 on JNa. To see if cAMP generation contributes to the effect of PGE2 on JNa, we tested the effect of exogenous cAMP, (8-chlorophenylthio(CPT)cAMP) on JNa. 0.1 mM 8-CPTcAMP reduced JNa from 35.75 +/- 2.3 to 21.6 +/- 2.2. However, the addition of PGE2 further blunted JNa to 15.9 +/- 1.3. In CCDs pretreated with indomethacin, 8-CPTcAMP did not significantly decrease JNa 33.6 +/- 2.8 vs. 28.4 +/- 2. However, superimposed PGE2 reduced JNa to 19.0 +/- 3.0. We conclude that PGE2 inhibits sodium transport predominantly by increasing intracellular calcium. This action is not mediated by a pertussis toxin-sensitive G protein. Finally, cAMP, through a cyclooxygenase-dependent mechanism, also inhibits CCD JNa and may contribute to the effects of PGE2 on JNa in the rabbit CCD.

Evidence for glomerular actions of epidermal growth factor in the rat.

Epidermal growth factor (EGF), an endogenous mitogenic peptide, has recently been shown to be a potent vasoconstrictor of vascular smooth muscle. In view of its potential role in proliferative and inflammatory renal glomerular diseases, we examined the effects of EGF both on cultured rat mesangial cells and on in vivo glomerular hemodynamics. Mesangial cells possess specific, saturable EGF receptors of differing affinities, with Kds of 0.1 and 1.7 nM, respectively. EGF produced a rapid increase in intracellular pH of 0.12 +/- 0.01 pH U, which was sodium dependent and amiloride inhibitable. The addition of EGF to mesangial cells cultured on either glass or dimethylpolysiloxane substratum induced reproducible cell contraction. Intrarenal EGF infusion did not affect systemic blood pressure or hematocrit but reversibly decreased GFR and renal blood flow from 4.19 +/- 0.33 to 3.33 +/- 0.26 and from 1.17 +/- 0.09 to 0.69 +/- 0.07 ml/min, respectively. Glomerular micropuncture confirmed decreases in single nephron plasma flow and in single nephron GFR (from 142 +/- 9 to 98 +/- 8 and from 51.6 +/- 11.7 to 28.5 +/- 3.5 nl/min, respectively) which were due to significant increases in both pre- and postglomerular arteriolar resistances (from 1.97 +/- 0.31 to 2.65 +/- 0.36 and from 1.19 +/- 0.11 to 2.00 +/- 0.15 10(10) dyn.s.cm-5 respectively) and to a significant decrease in the ultrafiltration coefficient, Kf, which fell from 0.100 +/- 0.019 to 0.031 +/- 0.007 nl/(s.mmHg). These studies demonstrate that mesangial cells possess specific receptors for EGF, and exposure of these cells to physiologic concentrations of EGF results in an in vitro functional response characterized by activation of Na+/H+ exchange and by resultant intracellular alkalinization, as well as by cell contraction. EGF administration in vivo significantly reduces the glomerular capillary ultrafiltration coefficient, Kf, which, in combination with EGF-induced constriction of both preglomerular and postglomerular arterioles, results in acute major reductions in the rates of glomerular filtration and perfusion.

Interactions of lysyl-bradykinin and antidiuretic hormone in the rabbit cortical collecting tubule.

Although intrarenal infusions of kinins produce diuresis, it is not clear to what extent this response is due to hemodynamically mediated medullary washout and/or to direct epithelial effects of kinins. Recent evidence has shown that bradykinin binds to collecting tubules in vitro. We therefore examined the interactions of lysyl-bradykinin and antidiuretic hormone (ADH) with respect to hydraulic conductivity (Lp) in the rabbit cortical collecting tubule perfused in vitro. To ensure adequate substrate for prostaglandin synthesis, the bath contained 2.5 microM arachidonic acid. Arachidonic acid produced no change in base-line Lp and had no effect on the subsequent response to a supramaximal dose of ADH (100 microU/ml). Therefore, all subsequent experiments were done in the presence of arachidonic acid. Lysyl-bradykinin (10(-9)M) added to either the lumen or bath had no effect on base-line Lp. Collecting tubules which were exposed for 1 h to bath lysyl-bradykinin (10(-9)M) had a significantly diminished subsequent Lp in response to ADH (P less than 0.02). In tubules exposed to bath lysyl-bradykinin plus indomethacin (5 microM), the subsequent ADH response was normal. Lysyl-bradykinin (10(-9)M) added to the lumen had no effect on subsequent ADH response. We conclude that lysyl-bradykinin from the basolateral side inhibits the hydroosmotic response of the cortical collecting tubule to ADH, and that this inhibition is probably prostaglandin-mediated. Lysyl-bradykinin does not affect water flow from the luminal surface. These data indicate that the diuresis seen with kinin infusions may result, at least in part, from a direct epithelial effect. They also suggest a role of the renal kallikrein-kinin system in modulating water transport in vivo.

Arachidonic acid epoxygenase Stereochemical analysis of the endogenous epoxyeicosatrienoic acids of human kidney cortex

Mass spectral and Chromatographic analysis demonstrates the presence of 14,15‐, 11,12‐ and 8,9‐epoxyeicosatrienoic acids (44%, 33% and 23% of the total, respectively) in human kidney cortex. Chiral analysis of the human renal expoxyeicosatrienoic acids shows the formation of 8,9‐, 11,12‐ and 14,15‐epoxyeicosatrienoic acids in a 1:1, 4:1 and 2:1 ratio of antipodes, respectively. These results demonstrate the biosynthetic origin of the human kidney 11,12‐ and 14,15‐epoxyeicosatrienoic acids and suggest a role for renal cytochrome P‐450 in the bioactivation of endogenous pools of arachidonic acid.

Cytochrome P450 metabolites of arachidonic acid are potent inhibitors of vasopressin action on rabbit cortical collecting duct.

AA is metabolized by a cytochrome P450, NADPH-dependent epoxygenase to four regioisomeric epoxyeicosatrienoic acids (EETs). The EETs are further hydrated enzymatically to their respective diols, vic-dihydroxyeicosatrienoic acids (DHETs). We studied the effect of pretreatment with DHETs on 10 microU/cm2 arginine vasopressin (AVP)-stimulated hydraulic conductivity (Lp) (Lp x 10(-7) cm/atm/s, mean +/- SE) in rabbit cortical collecting ducts (CCDs) perfused in vitro at 37 degrees C. At 10(-6) M all four DHETs were potent inhibitors of the hydroosmotic effect of AVP. 14,15-DHET was the most potent isomer; it reduced AVP-induced Lp from a control value of 234.75 +/- 11.7, n = 17, to a value of 95.2 +/- 8.39, n = 5, P less than 0.0001, a reduction of AVP-mediated water flow of 60%. The inhibitory effect of 14,15-DHET was dose dependent and significant to nanomolar concentrations. 14,15-DHET at 10(-7) M was as potent an inhibitor of AVPs activity as was 10(-7) M PGE2. AVPs hydroosmotic effect is mediated through its intracellular second messenger, cAMP. 8-p-Chlorophenylthio-cAMP (CcAMP) at 10(-4) M induced a peak Lp of 189.6 +/- 11.0, n = 8; pretreatment with 10(-6) M 14,15-DHET reduced CcAMP-peak Lp to 132.0 +/- 13.4, n = 5, P less than 0.01, demonstrating a post-cAMP effect. Gas chromatography/mass spectroscopy suggests that EETs are present in extracts purified from CCDs. We conclude that cytochrome P450 epoxygenase eicosanoids are potent inhibitors of the hydroosmotic effect of vasopressin and are endogenous constituents of normal CCDs, the major target tissue for AVP.

Mesangial deposition of type I collagen in human glomerulosclerosis

The presence of type I collagen in both diffuse and nodular diabetic glomerular lesions has been examined using immunohistochemical and electron microscopic techniques. At the ultrastructural level, banded collagen fibrils were observed in the mesangium in all cases of nodular (Kimmelstiel-Wilson) sclerosis and in 60% of the diffuse sclerotic lesions. Antibodies against type I collagen were localized in the fibrotic interstitium and the mesangium in all cases examined. Staining with type I collagen antibodies occurred in glomeruli with intact Bowmans capsules, and was predominantly localized to areas immediately adjacent to mesangial cells. In cases of focal sclerosis of nondiabetic origin, banded collagen fibrils and staining with anti-type I collagen antibody were observed in all cases in which the segmental lesion was presented in the specimen. The pattern of antibody localization in both the diabetic lesions and focal sclerosis differed from that obtained using anti-type IV (basement membrane) collagen antibodies. These results demonstrate that type I collagen is among the extracellular matrix components that comprise the sclerotic glomerular lesions of both diabetic and nondiabetic origin. Furthermore, the spatial localization of this collagen type suggests mesangial cell origin.