Stem cells in acute kidney injury repair.

New discoveries and developments in the biology and propagation of stem cells have fueled a whole new field of Medicine that has sparked great interest in the scientific community as well as in the general media and public. Stem cells are a very prominent and debated topic and have given ground to a new field of therapy in Medicine, which is called Regenerative Medicine. The kidney is a highly sophisticated and complicated organ, yet stem cell therapies have become of interest to Nephrologists and promising animal data are available showing the use of different stem cell populations in Nephrology. Acute kidney injury (AKI) is a clinical entity caused by a variety of factors resulting in renal damage and loss of function. Although it is reversible up to a point, it contributes tremendously to hospital morbidity and mortality. At the current time there are only supportive treatments available. Stem cell based therapies have the potential to become a broader and diverse treatment approach that is tailored to the pathophysiology of the disease and is therefore potentially more effective than traditional pharmacological approaches. The current article gives an overview about the field in general as well as potential treatment approaches and mechanisms.

The critical importance of urinary concentrating ability in the generation of urinary carbon dioxide tension.

Measurement of urine to blood (U-B) carbon dioxide tension (P(CO2)) gradient during alkalinization of the urine has been suggested to assess distal H(+) secretion. A fact that has not been considered in previous studies dealing with urinary P(CO2) is that dissolution of HCO(3) in water results in elevation of P(CO2) which is directly proportional to the HCO(3) concentration. To investigate the interrelationship of urinary HCO(3) and urinary acidification, we measured U-B P(CO2) in (a) the presence of enhanced H(+) secretion and decreased concentrating ability i.e., chronic renal failure (CRF), (b) animals with normal H(+) secretion and decreased concentrating ability, Brattleboro (BB) rats, and (c) the presence of both impaired H(+) secretion and concentrating ability (LiCl treatment and after release of unilateral ureteral obstruction). At moderately elevated plasma HCO(3) levels (30-40 meq/liter), normal rats achieved a highly alkaline urine (urine pH > 7.8) and raised urine HCO(3) concentration and U-B P(CO2). At similar plasma HCO(3) levels, BB rats had a much higher fractional water excretion and failed to raise urine pH, urine HCO(3) concentration, and U-B P(CO2) normally. At a very high plasma HCO(3) (>50 meq/liter), BB rats raised urine pH, urine HCO(3) concentration, and U-B P(CO2) to the same levels seen in normals. CRF rats failed to raise urine pH, urine HCO(3), and U-B P(CO2) normally at moderately elevated plasma HCO(3) levels; at very high plasma HCO(3) levels, CRF rats achieved a highly alkaline urine but failed to raise U-B P(CO2). Dogs and patients with CRF were also unable to raise urine pH, urine HCO(3) concentration, and U-B P(CO2) normally at moderately elevated plasma HCO(3) levels. In rats, dogs, and man, U-B P(CO2) was directly related to urine HCO(3) concentration and inversely related to fractional water excretion. At moderately elevated plasma HCO(3) levels, animals with a distal acidification defect failed to raise U-B P(CO2); increasing the plasma HCO(3) to very high levels resulted in a significant increase in urine HCO(3) concentration and U-B P(CO2). The observed urinary P(CO2) was very close to the P(CO2) which would be expected by simple dissolution of a comparable amount of HCO(3) in water. These data demonstrate that, in highly alkaline urine, urinary P(CO2) is largely determined by concentration of urinary HCO(3) and cannot be used as solely indicating distal H(+) secretion.

Evidence for cytokine-inducible nitric oxide synthesis from L-arginine in patients receiving interleukin-2 therapy.

An interferon-gamma, tumor necrosis factor, and interleukin-1-inducible, high-output pathway synthesizing nitric oxide (NO) from L-arginine was recently identified in rodents. High-dose interleukin-2 (IL-2) therapy is known to induce the same cytokines in patients with advanced cancer. Therefore, we examined renal cell carcinoma (RCC; n = 5) and malignant melanoma (MM; n = 7) patients for evidence of cytokine-inducible NO synthesis. Activity of this pathway was evaluated by measuring serum and urine nitrate (the stable degradation product of NO) during IL-2 therapy. IL-2 administration caused a striking increase in NO generation as reflected by serum nitrate levels (10- and 8-fold increase [P less than 0.001, P less than 0.003] for RCC and MM patients, respectively) and 24-h urinary nitrate excretion (6.5- and 9-fold increase [both P less than 0.001] for RCC and MM patients, respectively). IL-2-induced renal dysfunction made only a minor contribution to increased serum nitrate levels. Metabolic tracer studies using L-[guanidino-15N2]arginine demonstrated that the increased nitrate production was derived from a terminal guanidino nitrogen atom of L-arginine. Our results showing increased endogenous nitrate synthesis in patients receiving IL-2 demonstrate for the first time that a cytokine-inducible, high-output L-arginine/NO pathway exists in humans.

Effects of triiodothyronine replacement on the anemia of chronic renal failure.

Patients and/or experimental animals with chronic renal failure have decreased serum levels of triiodothyronine (T3), a hormone well known for its erythropoietic activity. The following studies were designed in order to determine whether this observed abnormality in T3 metabolism is an important contributory factor to the etiology of the anemia of uremia. Groups of rats were made chronically uremic by a standard 5/6 nephrectomy technique and received slightly above physiological doses of T3 either by intermittent S.C. injections (twice daily) or by continuous infusion from intraperitoneally implanted osmotic minipumps. After 2 weeks of such treatment, and despite a normalization of serum T3 levels, there were no significant changes in the hematocrit, individual red cell mass, or serum erythropoietin levels of the uremic animals given T3 compared to control rats. We conclude that (1) the decreased serum T3 levels observed in uremia are not an important contributory factor to the pathogenesis of the anemia, and (2) treatment with replacement doses of T3 does not result in significant improvement of erythropoiesis.

Metolazone and spironolactone in cirrhosis and the nephrotic syndrome.

Eighteen patients with hepatic cirrhosis or nephrotic syndrome and having edema and/or ascites were treated during successive periods with metolazone 5 to 40 mg/day, spironolactone 100 mg/day, and with both diuretics concurrently. Metolazone alone produced a marked diuresis, natriuresis, and weight loss in 8 patients. Spironolactone alone had little effect, but the addition of metolazone renewed diuresis and natriuresis and resulted in additional substantial weight losses in all patients responsive to metolazone alone. Concurrent spironolactone and metolazone also induced moderate diuretic effects in some patients who failed to respond to either drug alone. The drugs were well tolerated; the administration of spironolactone with metolazone prevented decreases in serum potassium, which had occurred during treatment with metolazone alone.

Secretion of atrial natriuretic peptide (ANP) from fish atrial and ventricular myocytes in tissue culture.

Primary cultures of atrial and ventricular myocytes (approx. 1 x 10(5) cells/culture) were prepared from adult teleost fish Gila atraria and maintained for 10 days. Immunoreactive atrial natriuretic peptide (ir-ANP) from fish atrial and ventricular cells was 3.9 and 2.8 ng/culture respectively, values not significantly different. Atriocytes from rat and mouse secreted comparable amounts of ANP which were not significantly different from atrial fish cultures (5.2 and 4.3 ng/culture). In contrast, their ventricular myocytes secreted only small quantities of ANP (0.8 and 0.3 ng/culture). When analyzed by reversed-phase HPLC, the media of both fish atrial and ventricular myocytes contained a peptide which exhibited properties similar to authentic human ANP (Ser 99-Tyr 126), suggesting a significant degree of sequence homology between fish and mammalian ANP. Fish ventricular cells, unlike normal mammalian ventricular cells, secrete substantial quantities of immunoreactive-ANP.

Effect of vanadate on water transport by the toad bladder.

Vanadate increases renal Na and water excretion. The mechanism whereby vanadate impairs water transport was examined in the toad bladder. Vanadate did not alter baseline water transport but caused a significant inhibition of water transport elicited by high doses of AVP. The inhibition of AVP stimulated water flow by vanadate was dose dependent with inhibition present with concentration as low as 10(-7) and maximal inhibition occurring at 10(-5) M. Vanadate also inhibited water transport stimulated by cyclic AMP or by phosphodiesterase inhibition indicating that vanadate has an effect beyond cyclic AMP step, in addition to whatever effect it might have on adenylate cyclase. The inhibitory effect of vanadate on AVP stimulated water flow was not altered by prior Na-K-ATPase or prostaglandin inhibition. Since vanadate has been shown to stimulate adenylate cyclase in other tissues we examined whether addition of vanadate 10 minutes after addition of AVP would enhance water transport. Vanadate caused a transient enhancement of AVP stimulated water flow. These data demonstrate that vanadate can inhibit or stimulate water flow in the toad bladder.

In vivo renal angiotensin converting enzyme activity decreases in glycerol-induced acute renal failure.

We previously demonstrated that intrarenal angiotensin II generation during glycerol-induced acute renal failure was attenuated, which may have resulted from the inability of intrarenal converting enzyme to convert renal angiotensin I to angiotensin II. In order to test this hypothesis in vivo, we determined the ability of the kidney to convert angiotensin I to angiotensin II by measuring the decrease in renal cortical blood flow (RCBF) in response to exogenous angiotensin I administration. Changes in RCBF were monitored by laser-Doppler velocimetry. Three groups of rats were studied: Group I, controls (N = 7); 24 hours prior to study Group II animals were injected with 50% glycerol, 8 ml/kg i.m. (N = 4); and Group III rats were injected with mercuric chloride, 3 mg/kg s.c. (N = 5). All experimental animals had a three- to sixfold rise in serum creatinine. Mean glomerular filtration rate (GFR) of the left and right kidney in control rats was 0.7 and 0.7 ml/min, respectively. Twenty-four hours after glycerol, GFR was 0.2 ml/min in the left kidney and 0.2 ml/min in the right kidney. In HgCl2 treated rats GFR was 0.1 ml/min in the left kidney and 0.1 ml/min in the right kidney. Each of the following maneuvers elicited a similar rise in blood pressure in Groups I through III. Specifically, when first angiotensin I (4 micrograms/kg/min) was infused for three minutes; second, when 10 minutes later angiotensin I (5 micrograms) was directly applied on the left kidney; and third, when angiotensin II (5 micrograms) was topically administered.(ABSTRACT TRUNCATED AT 250 WORDS)

Estimation of renal interstitial adenosine and purine metabolites by microdialysis.

It has been proposed that adenosine, derived from ATP and released into the renal interstitium, mediates a reduction in renal function in ischemic acute renal failure. Because no direct measurements of interstitial adenosine are available, we evaluated an in vivo microdialysis technique to assess the levels of adenosine and its metabolites in the cortex of the normal rat kidney (n = 6). Microdialysis probe implantation did not alter cortical renal blood flow, glomerular filtration rate, or fractional sodium excretion. The interstitial concentration of adenosine was 199 +/- 53 nM, and relative concentrations of inosine, hypoxanthine, xanthine, and uric acid were 99 +/- 47, 182 +/- 29, and 183 +/- 70 nM and 1.8 +/- 0.4 microM, respectively. Infusion of ATP-MgCl2 (n = 5) resulted in a significant increase in the dialysate levels of adenosine (67 +/- 11 to 378 +/- 97 nM), inosine (230 +/- 102 to 803 +/- 219 nM), and uric acid (3.5 +/- 1.3 to 6.9 +/- 1.7 microM). In conclusion, this study demonstrates that the microdialysis technique is suited to monitor metabolically important substances in the renal interstitium.

Urea induces the heat shock response in human neuroblastoma cells.

Uremic encephalopathy is a complication of renal failure that reflects stresses exerted by as yet poorly defined uremic toxins. All cells respond to stresses by undergoing the "heat shock" response. Although urea kinetics and creatinine concentration are routinely used to assess dialysis adequacy, the roles of urea and creatinine as uremic toxins remain controversial. To investigate their potential roles in uremic encephalopathy, cultured human neuroblastoma cells (SK-N-SH) were exposed to 0.5 to 14 mg/dL creatinine, or to 20 to 200 mg/dL urea, or to mannitol, NaCl, or glycerol at equivalent osmolalities for 30 min to 48 h, and the induction of Hsp72 (heat shock) protein was used as a marker of cell stress. Although creatinine failed to elicit a heat shock response, urea in clinically relevant concentrations (40 to 200 mg/dL) induced it at 30 min. The response peaked at 10 h and returned to zero by 48 h. Cells exposed to equivalent osmolalities of mannitol, NaCl, or glycerol failed to exhibit this response. Protein extracts from cells exposed to urea showed significant carbamylation that increased as a function of time. These results demonstrate: (1) that urea is neurotoxic in vitro and that creatinine is not: (2) that the insult urea causes is not simply the result of hypertonicity; but rather (3) that urea, via breakdown to cyanate and ammonium ions, may cause cell stress because of its ability to cause carbamylation of cellular proteins. The cells attenuation of the heat shock response after 10 h of exposure to urea suggests that they can adapt to the presence of urea or carbamylation. This may explain, in part, why the same degree of azotemia causes fewer neurological symptoms in patients with chronic as opposed to acute renal failure.

Erythropoietin stimulates proliferation of human renal carcinoma cells.

BACKGROUND: We reported recently that normal human, rat, and mouse tubular cells express authentic erythropoietin-receptors (EPO-R) through which EPO stimulates mitogenesis. The present study examines whether EPO could elicit such a proliferative and thereby potentially detrimental response in cells of human renal-cell carcinoma (RCC). METHODS: Nephrectomy samples were screened from patients with RCC (one chromophilic, two clear cell) as well as cell lines of human (Caki-2, 786-0) and mouse (RAG) renal adenocarcinomas for expression of EPO-R transcripts and protein. Cells were further tested for specific 125I-EPO binding and mitogenic response to EPO. RESULTS: Authentic EPO-R transcripts and protein (approximately 72 kD) were detected in renal tumors and cell lines. Tumors showed low-level EPO expression, while cell lines did not. In cells, specific 125I-EPO binding to a single class of EPO-R (apparent Kd 1. 3 to 1.4 nmol/L, Bmax 2.2 to 2.6 fmol/mg protein) was observed. EPO stimulated cell proliferation dose dependently, and the individual mitogenic effects of either EPO or 10% newborn calf serum were markedly amplified when both were coadministered. CONCLUSION: These data are the first to demonstrate, to our knowledge, that human RCCs express EPO-R message and protein and that receptor activation stimulates their proliferation in vitro. If these mitogenic effects of EPO are also operative in patients with RCC, endogenous EPO or its administration for the treatment of anemia could potentially hasten proliferation of renocellular malignancies.

The kidney in hypergammaglobulinemic purpura.

A 25-year-old woman with long-standing hypergammaglobulinemic purpura developed distal renal tubular acidosis and a urine-concentrating defect. The acidification defect was characterized as suggestive of impaired distal proton secretion by infusion of neutral phosphate. The concentrating defect was a form of acquired nephrogenic diabetes insipidus. On renal biopsy, IgM mesangial nephropathy was found along with multiple large hyaline tubular casts. The renal findings in hypergammaglobulinemic purpura are reviewed.

Modification of Na and Cl transport in canine tracheal mucosa by prostaglandins.

We tested the effect of prostaglandins PGF2 alpha and PGE1 on the transport of 36Cl and 22Na by canine tracheal epithelium. Sheets of epithelium were mounted in Ussing chambers and short-circuited. Addition of PGF2 alpha to the mucosal side resulted in an increase of net Cl secretion from 0.71 +/- 0.41 to 2.40 +/- 0.67 mu eq . cm-2 . h-1 without significant effect on net Na absorption. Prostaglandin E1 on the mucosal side increased net Cl secretion from 1.36 +/- 0.31 to 2.69 +/- 0.35 and decreased Na absorption from 0.87 +/- 0.16 to 0.49 +/- 0.09. Indomethacin significantly depressed net Cl secretion from 1.36 +/- 0.36 to 0.57 +/- 0.22. Subsequent addition of PGE1 augmented net Cl secretion to 3.88 +/- 0.75. PGE1 did not enhance [14C]mannitol fluxes across this epithelium. Cellular levels of cAMP increased in response to PGE1 from 130 +/- 12.7 to 642 +/- 33.4 pmol . mg prot-1 . 10 min-1, whereas PGF2 alpha had no effect. These data suggest that although effects of PGF2 alpha and PGE1 are similar as pertains to net Cl secretion, they differ in their effects on Na transport and their capacity to increase cAMP levels. Alterations in Cl and Na transport in response to PGE1 are likely to be mediated, at least in part, by the adenylate cyclase-cAMP system. Furthermore, endogenous prostaglandins may have an important regulatory role in ion transport by airways epithelium.

Effect of vanadate on renal tubular function in rats.

Orthovanadate (VO4) has been shown to cause a marked natriuresis in rats. This has been ascribed to its inhibitory action on renal Na-K-ATPase activity. Because virtually all nephron segments possess Na-K-ATPase activity the administration of VO4 should alter renal tubular transport along the entire nephron. To examine this possibility, adult rats were anesthetized and infused with VO4 (10 mumol.kg body wt-1.h-1 i.v.). This dose had no effect on glomerular filtration rate, effective renal plasma flow, and blood pressure, whereas urine flow and sodium and water excretion rose markedly. Potassium excretion remained unaltered. VO4 depressed only maximal bicarbonate and glucose reabsorption without causing a glucose or bicarbonate "leak" at normal levels of blood glucose or bicarbonate. In acutely thyroparathyroidectomized rats VO4 produced a striking phosphaturia, not accompanied by an increase in nephrogenous cAMP excretion. Both free water clearance in Brattleboro rats and free water reabsorption in normal rats was significantly depressed by VO4. These data demonstrate that VO4 depresses tubular reabsorption in proximal and distal nephron segments. We conclude that VO4 exerts its effect on tubular function by inhibition of Na-K-ATPase activity.

Serosal Na/Ca exchange and H+ and Na+ transport by the turtle and toad bladders.

A Na/Ca exchange system has been described in the plasma membrane of several tissues and seems to regulate the concentration of calcium in cytosol. Replacement of extracellular Na by sucrose increases calcium uptake into and decreases calcium efflux from the cell, leading to an increase in cytosolic calcium. The effect of an increase in cytosolic calcium mediated by the Na/Ca exchange system on H+ and Na transport in the turtle and toad bladder was investigated by replacing serosal Na isosmotically by sucrose or choline. Replacement of serosal by sucrose was associated with a significant inhibition of H+ secretion or Na transport which was reversible by addition of NaCl. Replacement of mucosal Na by sucrose failed to alter H+ secretion. Removal of serosal Na was associated with a significant increase in 45Ca uptake which could be blocked by pretreatment with lanthanum chloride. Pretreatment with lanthanum chloride blunted the inhibitory effect of replacement of serosal Na by sucrose on H+ and Na transport, thus suggesting that the increase in calcium uptake and the inhibition of transport are causally related. Under anaerobic conditions the rate of H+ or Na transport are linked to the rate of lactate production. The inhibition of Na or H+ transport by removal of serosal Na was accompanied by a proportional decrease in lactate production, thus suggesting that an increase in cytosolic calcium does not inhibit transport by uncoupling glycolysis from transport. Replacement of serosal Na by sucrose did not alter the force of the H+ or Na pump but led to an increase in resistance of the active pathway of H+ and Na transport. The inhibition of Na transport by replacement of serosal Na with sucrose could be reversed by addition of amphotericin B, an agent which increases luminal permeability to Na, thus suggesting that decreased Na entry across the apical membrane is the mechanism responsible for the inhibition of Na transport. The results of the present studies strongly suggest that an increase in cytosolic calcium through the serosal Na/Ca exchange system inhibits H+ and Na transport in the turtle and toad bladder probably by increasing the resistance of the luminal membrane.

Characterization of Na-K-ATPase in dog tracheal epithelium: enzymatic and ion transport measurements.

The dog tracheal epithelium actively secretes Cl and absorbs Na. The possible dependency of this electrolyte transport on a Mg-dependent, Na-K-activated adenosine triphosphatase (Na-K-ATPase, EC 3.6.1.3) was examined. The characteristics of this enzyme system were investigated using homogenates of tracheal epithelium. The electrical properties and ion fluxes of this epithelium were determined in tissues mounted in Ussing chambers. Addition of Na and K produced an approximate 50% activation of basal Mg-ATPase activity. The apparent Km values for ATP, Na, K, and Mg were 0.4, 12.7, 1.9, and 1.6 mM, respectively. The total specific ATPase activity was 8.1 +/- 0.4 and that of the Mg-ATPase 4.3 +/- 0.1 mumol Pi. mg protein -1.h-1. Addition of ouabain (1 muM) or omission of K from the submucosal bathing solution reduced potential difference (PD) and short-circuit current (SCC) significantly. Relatively low concentrations (0.1 mM or less) of ethacrynic acid, furosemide, or 2,4-dinitrophenol (2,4-DNP) depressed SCC and PD significantly, i.e., at concentrations that were without effect on the Na-K-ATPase activity. Ethacrynic acid inhibited Cl secretion, whereas 2,4-DNP lowered both Na and Cl transport. These data demonstrate that 1) the tracheal mucosa of dogs contains a Na-K-ATPase at relatively high specific activity, 2) this enzyme is likely contained in the basal aspect of this membrane, 3) it appears to be essential for maintenance of Cl secretion, and 4) Cl secretion can be reduced (by ethacrynic acid, furosemide, and 2,4-DNP) without Na-K-ATPase inhibition.

Human, rat, and mouse kidney cells express functional erythropoietin receptors.

BACKGROUND: Erythropoietin (EPO), secreted by fibroblast-like cells in the renal interstitium, controls erythropoiesis by regulating the survival, proliferation, and differentiation of erythroid progenitor cells. We examined whether renal cells that are exposed to EPO express EPO receptors (EPO-R) through which analogous cytokine responses might be elicited. METHODS: Normal human and rat kidney tissue and defined cell lines of human, rat, and mouse kidney were screened, using reverse transcription-polymerase chain reaction, nucleotide sequencing, ligand binding, and Western blotting, for the expression of EPO-R. EPO's effects on DNA synthesis and cell proliferation were also examined. RESULTS: EPO-R transcripts were readily detected in cortex, medulla, and papilla of human and rat kidney, in mesangial (human, rat), proximal tubular (human, mouse), and medullary collecting duct cells (human). Nucleotide sequences of EPO-R cDNAs from renal cells were identical to those of erythroid precursor cells. Specific 125I-EPO binding revealed a single class of high- to intermediate-affinity EPO-Rs in each tested cell line (kD 96 pm to 1. 4 nm; Bmax 0.3 to 7.0 fmol/mg protein). Western blots of murine proximal tubular cell membranes revealed an EPO-R protein of approximately 68 kDa. EPO stimulated DNA synthesis and cell proliferation dose dependently. CONCLUSION: This is the first direct demonstration, to our knowledge, that renal cells possess EPO-Rs through which EPO stimulates mitogenesis. This suggests currently unrecognized cytokine functions for EPO in the kidney, which may prove beneficial in the repair of an injured kidney while being potentially detrimental in renal malignancies.

Correlation of creatinine levels in saliva and plasma in normal subjects and renal patients.

Whole saliva and serum creatinine levels were evaluated in three normal subjects and 12 patients with chronic renal failure receiving regular hemodialysis treatments. Ratios of the serum vs. saliva creatinine levels ranging from 4.5 to 30.0 were found. Repeated studies also revealed marked intra-subject variations in these ratios. We conclude that the monitoring of whole saliva creatinine levels is only of limited value in accurately predicting serum or plasma creatinine levels, despite its appeal of simplicity, convenience and non-invasiveness. Its value in the qualitative monitoring of renal function may, however, be useful.