American Journal of Physiology - Renal Physiology

Elucidation of the gene defects responsible for many disorders of renal fluid and electrolyte homeostasis has provided new insights into normal and abnormal physiology. Identifying the causes of Gitelman's and Bartter's syndromes has greatly enhanced our understanding of ion transport by thick ascending limb and distal convoluted tubule cells. Despite this information, several phenotypic features of these diseases remain confusing, even in the face of molecular insight. Paramount among these are disorders of divalent cation homeostasis. Bartter's syndrome is caused by dysfunction of thick ascending limb cells. It is associated with calcium wasting, but magnesium wasting is usually mild. Loop diuretics, which inhibit ion transport by thick ascending limb cells, markedly increase urinary excretion of both calcium and magnesium. In contrast, Gitelman's syndrome is caused by dysfunction of the distal convoluted tubule. Hypocalciuria and hypomagnesemia are universal parts of this disorder. Yet although thiazide diuretics, which inhibit ion transport by distal convoluted tubule cells, reduce urinary calcium excretion, they have minimal effects on urinary magnesium excretion, when given acutely. This review proposes mechanisms that may account for the differences between the effects of diuretic drugs and the phenotypic features of Gitelman's and Bartter's syndromes. These mechanisms are based on recent insights from another inherited disease of ion transport, inherited magnesium wasting, and from a review of the chronic effects of diuretic drugs in animals and people.

Renal tubular epithelial cell (RTC) apoptosis causes tubular atrophy, a hallmark of renal disease progression. Apoptosis is generally characterized by reduced cell volume and cytosolic pH, but epithelial cells are relatively resistant to shrinkage due to regulatory volume increase, which is mediated by Na⁺/H⁺exchanger (NHE) 1. We investigated whether RTC apoptosis requires caspase cleavage of NHE1. Staurosporine- and hypertonic NaCl-induced RTC apoptosis was associated with cell shrinkage and diminished cytosolic pH, and apoptosis was potentiated by amiloride analogs, suggesting NHE1 activity opposes apoptosis. NHE1-deficient fibroblasts demonstrated increased susceptibility to apoptosis, which was reversed by NHE1 reconstitution. NHE1 expression was markedly decreased in apoptotic RTC due to degradation, and preincubation with peptide caspase antagonists restored NHE1 expression, indicating that NHE1 is degraded by caspases. Recombinant caspase-3 cleaved the in vitro-translated NHE1 cytoplasmic domain into five distinct peptides, identical in molecular weight to NHE1 degradation products derived from staurosporine-stimulated RTC lysates. In vivo, NHE1 loss-of-function C57BL/6.SJL- swe/swemice with adriamycin-induced nephropathy demonstrated increased RTC apoptosis compared with adriamycin-treated wild-type controls, thereby implicating NHE1 inactivation as a potential mechanism of tubular atrophy. We conclude that NHE1 activity is critical for RTC survival after injury and that caspase cleavage of RTC NHE1 may promote apoptosis and tubular atrophy by preventing compensatory intracellular volume and pH regulation.

Na⁺/H⁺exchanger-1 (NHE1) is a ubiquitous plasma membrane Na⁺/H⁺exchanger typically associated with maintenance of intracellular volume and pH. In addition to the NHE1 role in electroneutral Na⁺/H⁺transport, in renal tubular epithelial cells in vitro the polybasic, juxtamembrane NHE1 cytosolic tail domain acts as a scaffold, by binding with ezrin/radixin/moesin (ERM) proteins and phosphatidylinositol 4,5-bisphosphate, which initiates formation of a signaling complex that culminates in Akt activation and opposition to initial apoptotic stress. With robust apoptotic stimuli renal tubular epithelial cell NHE1 is a caspase substrate, and proteolytic cleavage may permit progression to apoptotic cell death. In vivo, genetic or pharmacological NHE1 loss of function causes renal tubule epithelial cell apoptosis and renal dysfunction following streptozotocin-induced diabetes, ureteral obstruction, and adriamycin-induced podocyte toxicity. Taken together, substantial in vivo and in vitro data demonstrate that NHE1 regulates tubular epithelial cell survival. In contrast to connotations of NHE1 as an unimportant “housekeeping” protein, this review highlights that NHE1 activity is critical for countering tubular atrophy and chronic renal disease progression.

Recent investigations demonstrate increased Na/H exchanger-1 (NHE-1) activity and plasma levels of ouabain-like factor in spontaneously hypertensive rats. At nanomolar concentrations, ouabain increases Na-K-ATPase activity, induces cell proliferation, and activates complex signaling cascades. We hypothesize that the activity of NHE-1 and Na-K-ATPase are interdependent. To test whether treatment with picomolar ouabain regulates Na-K-ATPase through an NHE-1-dependent mechanism, we examined the role of NHE-1 in ouabain-mediated stimulation of Na-K-ATPase in kidney proximal tubule cell lines [opossum kidney (OK), HK-2, HKC-5, and HKC-11] and rat kidney basolateral membranes. Ouabain stimulated Na-K-ATPase activity and tyrosine phosphorylation in cells that express NHE-1 (OK, HKC-5, and HKC-11) but not in HK-2 cells that express very low levels of NHE-1. Inhibition of NHE-1 with 5 μM EIPA, a NHE-1-specific inhibitor, prevented ouabain-mediated stimulation of⁸⁶Rb uptake and Na-K-ATPase phosphorylation in OK, HKC-5, and HKC-11 cells. Expression of wild-type NHE-1 in HK2 cells restored regulation of Na-K-ATPase by picomolar ouabain. Treatment with picomolar ouabain increased membrane expression of Na-K-ATPase and enhanced NHE-1-Na-K-ATPase α1-subunit association. Treatment with ouabain (1 μg·kg body wt⁻¹·day⁻¹) increased Na-K-ATPase activity, expression, phosphorylation, and association with NHE-1 increased in rat kidney cortical basolateral membranes. Eight days' treatment with ouabain (1 μg·kg body wt⁻¹·day⁻¹) resulted in increased blood pressure in these rats. These results suggest that the association of NHE-1 with Na-K-ATPase is critical for ouabain-mediated regulation of Na-K-ATPase and that these effects may play a role in cardioglycoside-stimulated hypertension.

Parathyroid hormone (PTH) inhibits Na⁺-K⁺-ATPase activity by serine phosphorylation of the α₁-subunit through ERK-dependent phosphorylation and translocation of protein kinase Cα (PKCα). On the basis of previous studies, we postulated that PTH regulates sodium pump activity through Src kinase, PLC, and calcium-dependent ERK phosphorylation. In the present work utilizing opossum kidney cells, a model of renal proximal tubule, PTH-stimulated ERK phosphorylation and membrane translocation of PKCα were prevented by inhibition of Src kinase, PLC, and calcium entry. Pharmacological inhibition of PLA₂ did not prevent PTH-stimulated ERK phosphorylation but completely prevented PKCα translocation. Silencing the expression of cytosolic or calcium-independent PLA₂ also prevented PTH-mediated phosphorylation of Na⁺-K⁺-ATPase α₁-subunit and PKCα without blocking ERK phosphorylation. Inhibition of Na⁺-K⁺-ATPase activity by the PLA₂ metabolites arachidonic acid and 20-hydroxyeicosatetraenoic acid was prevented by specific inhibition of PKCα but not by U0126, a MEK-1 inhibitor. Transient transfection of constitutively active MEK-1 cDNA induced phosphorylation of Na⁺-K⁺-ATPase α₁-subunit and PKCα, which was prevented by PLA₂ inhibition. We conclude that PTH stimulates Na⁺-K⁺-ATPase phosphorylation and decreases the activity of Na⁺-K⁺-ATPase by a sequential activation of a signaling pathway involving Src kinase, PLC, ERK, PLA₂, and PKCα.

Patients with end-stage kidney disease on dialysis have increased mortality and reduced physical activity, contributing to impaired physical function. Although exercise programs have demonstrated a positive effect on physiological outcomes such as cardiovascular function and strength, there is a reduced focus on physical function. The aim of this review was to determine whether exercise programs improve objective measures of physical function indicative of activities of daily living for patients with end-stage kidney disease on dialysis. A systematic search of Medline, Embase, the Cochrane Central Register of Controlled Trials, and Cumulative Index to Nursing and Allied Health Literature identified 27 randomized control trials. Only randomized control trials using an exercise intervention or significant muscular activation in the intervention, a usual care, nonexercising control group, and at least one objective measure of physical function were included. Participants were ≥18 yr of age, with end-stage kidney disease, undergoing hemo- or peritoneal dialysis. Systematic review of the literature and quality assessment of the included studies used the Cochrane Collaboration’s tool for assessing risk bias. A meta-analysis was completed for the 6-min walk test. Data from 27 studies with 1,156 participants showed that exercise, regardless of modality, generally increased 6-min walk test distance, sit-to-stand time or repetitions, and grip strength as well as step and stair climb times or repetitions, dynamic mobility, and short physical performance battery scores. From the evidence available, exercise, regardless of modality, improved objective measures of physical function for end-stage kidney disease patients undergoing dialysis. It is acknowledged that further well-designed randomized control trials are required.

We have previously described a cell surface channel complex that is highly selective for nucleic acid ( 6 , 7 ). The channel complex was purified to homogeneity by solubilizing renal brush-border membranes (BBM) with CHAPS and separation by liquid chromatography. It was characterized by reconstitution in planar lipid bilayers. The channel consists of a pore-forming subunit that is blocked by heparan sulfate and a regulatory subunit that is blocked by l-malate ( 7 ). The current studies were performed to compare the characteristics of the nucleic acid-conducting channel in native BBM with the characteristics that have been determined for the complex reconstituted from purified proteins. BBM were purified by differential centrifugation and reconstituted in lipid bilayers. Current was not observed until oligodeoxynucleotide (ODN) was added. Conductance was 9.1 ± 0.9 pS; rectification and voltage dependence were not observed. Reversal potential ( E_rev) shifted to +14 ± 0.1 mV by a 10-fold gradient for ODN but was not altered when gradients were created for any other ion. Open probability increased significantly with an increase in Ca²⁺on the trans chamber of the bilayer apparatus. Changes in cis Ca²⁺were without effect. Addition of l-malate to the cis chamber or heparan sulfate to the trans chamber significantly reduced the open probability of the channel. These data demonstrate that the nucleic acid channel in BBM is electrophysiologically and pharmacologically identical to that previously reported for purified protein and demonstrate that a nucleic acid-conducting channel is a component of renal BBM.

Escherichia coli and Staphylococcus typhimurium are known to accumulate betaine by increased transport when extracellular osmolality rises. In the present studies a similar process is demonstrated in mammalian cells. Renal medullary cells contain high concentrations of "compatible" organic osmolytes such as betaine, myo-inositol, sorbitol, and glycerophosphorylcholine. The organic osmolytes occur as an osmoregulatory response to the high and variable interstitial NaCl concentration that is part of the urinary concentrating mechanism. Dog kidney cells in culture (MDCK) were previously shown to accumulate betaine in response to increased extracellular osmolality. We demonstrate here that this accumulation requires the presence of betaine in the medium, and this apparently is a result of uptake of extracellular betaine, rather than synthesis by the cells. MDCK cells have low- and high-affinity sodium-dependent betaine transporters with Km for betaine of approximately 6 and approximately 0.1 mM, respectively. Relative to isotonic controls, sodium-dependent betaine uptake is approximately sevenfold greater in cells chronically exposed (greater than 1 yr) to hypertonic medium (615 mosmol/kg). This is due to an increase in the maximal velocity of sodium-dependent betaine uptake with no apparent change in Km. Cells acutely exposed (1-7 days) to hypertonic medium show increased sodium-dependent betaine uptake, which is maximal after 1 day, then decreases as betaine and other osmolytes accumulate in the cells. Thus the response by which renal cells accumulate betaine following hypertonicity apparently includes an increase in the number (or, less likely, the transport turnover rate) of functioning sodium-dependent betaine transporters.

During systemic acidosis, renal proximal tubular cells exhibit enhanced rates of bicarbonate and ammonium ion synthesis and undergo extensive hypertrophy. The former adaptations are accomplished, in part, by increased expression of glutaminase (GA). LLC-PK₁-FBPase⁺cells, a gluconeogenic line of porcine kidney cells, exhibit a rapid activation of the ERK1/2 and p38 MAPK pathways and a two- to threefold increase in GA mRNA when transferred to acidic medium (pH 6.9). Transforming growth factor-β (TGF-β), a potent activator of MAPK and Smad signaling cascades, also causes extensive renal hypertrophy. Thus the potential role of TGF-β in the renal response to metabolic acidosis was investigated. Western blot analyses established that in LLC-PK₁-FBPase⁺cells, TGF-β activated the ERK1/2, p38 MAPK, and Smad1/5/8 pathways, but not the JNK and Smad2/3 pathways. Addition of TGF-β to cells cultured in normal medium (pH 7.4) produced a steady increase in GA mRNA, resulting in a twofold induction after 18 h. Western blot analysis indicated that treatment with either TGF-β or acidic medium resulted in an increased level of fibronectin. However, the effects of the two treatments on both GA mRNA and fibronectin levels occurred with different time courses and were additive. In addition, the rates of ammonia production were decreased slightly by addition of TGF-β. Finally, a GA-luciferase reporter construct, which is activated 3.5-fold by treatment with acidic medium, is not affected by TGF-β. Therefore, TGF-β and metabolic acidosis activate some of the same signaling pathways in LLC-PK₁-FBPase⁺cells, but produce separate effects on GA expression.

Pericytes are tissue-resident mesenchymal progenitor cells anatomically associated with the vasculature that have been shown to participate in tissue regeneration. Here, we tested the hypothesis that kidney pericytes, derived from FoxD1⁺ mesodermal progenitors during embryogenesis, are necessary for postnatal kidney homeostasis. Diphtheria toxin delivery to FoxD1Cre::RsDTR transgenic mice resulted in selective ablation of >90% of kidney pericytes but not other cell lineages. Abrupt increases in plasma creatinine, blood urea nitrogen, and albuminuria within 96 h indicated acute kidney injury in pericyte-ablated mice. Loss of pericytes led to a rapid accumulation of neutral lipid vacuoles, swollen mitochondria, and apoptosis in tubular epithelial cells. Pericyte ablation led to endothelial cell swelling, reduced expression of vascular homeostasis markers, and peritubular capillary loss. Despite the observed injury, no signs of the acute inflammatory response were observed. Pathway enrichment analysis of genes expressed in kidney pericytes in vivo identified basement membrane proteins, angiogenic factors, and factors regulating vascular tone as major regulators of vascular function. Using novel microphysiological devices, we recapitulated human kidney peritubular capillaries coated with pericytes and showed that pericytes regulate permeability, basement membrane deposition, and microvascular tone. These findings suggest that through the active support of the microvasculature, pericytes are essential to adult kidney homeostasis.