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The distal nephron is a heterogeneous part of the nephron composed by six different cell types, forming the epithelium of the distal convoluted (DCT), connecting, and collecting duct. To dissect the function of these cells, knockout models specific for their unique cell marker have been created. However, since this part of the nephron develops at the border between the ureteric bud and the metanephric mesenchyme, the specificity of the single cell markers has been recently questioned. Here, by mapping the fate of the aquaporin 2 (AQP2) and Na+-Cl−cotransporter (NCC)-positive cells using transgenic mouse lines expressing the yellow fluorescent protein fluorescent marker, we showed that the origin of the distal nephron is extremely composite. Indeed, AQP2-expressing precursor results give rise not only to the principal cells, but also to some of the A- and B-type intercalated cells and even to cells of the DCT. On the other hand, some principal cells and B-type intercalated cells can develop from NCC-expressing precursors. In conclusion, these results demonstrate that the origin of different cell types in the distal nephron is not as clearly defined as originally thought. Importantly, they highlight the fact that knocking out a gene encoding for a selective functional marker in the adult does not guarantee cell specificity during the overall kidney development. Tools allowing not only cell-specific but also time-controlled recombination will be useful in this sense.

A low-Na+, high-K+diet (LNaHK) is considered a healthier alternative to the “Western” high-Na+diet. Because the mechanism for K+secretion involves Na+reabsorptive exchange for secreted K+in the distal nephron, it is not understood how K+is eliminated with such low Na+intake. Animals on a LNaHK diet produce an alkaline load, high urinary flows, and markedly elevated plasma ANG II and aldosterone levels to maintain their K+balance. Recent studies have revealed a potential mechanism involving the actions of alkalosis, urinary flow, elevated ANG II, and aldosterone on two types of K+channels, renal outer medullary K+and large-conductance K+channels, located in principal and intercalated cells. Here, we review these recent advances.

The aldosterone-sensitive distal nephron (ASDN) exhibits axial heterogeneity in structure and function from the distal convoluted tubule to the medullary collecting duct. Ion and water transport is primarily divided between the cortex and medulla of the ASDN, respectively. Transcellular transport in this segment is highly regulated in health and disease and is integrated across different cell types. We currently lack an inexpensive, high-yield, and tractable technique to harvest and culture cells for the study of gene expression and physiological properties of mouse cortical ASDN. To address this need, we harvested tubules bound to Dolichos biflorus agglutinin lectin-coated magnetic beads from the kidney cortex and characterized these cell preparations. We determined that these cells are enriched for markers of distal convoluted tubule, connecting tubule, and cortical collecting duct, including principal and intercalated cells. In primary culture, these cells develop polarized monolayers with high resistance (1,000-1,500 Ω * cm2) and maintain expression and activity of key channels. These cells demonstrate an amiloride-sensitive short-circuit current that can be enhanced with aldosterone and maintain measurable potassium and anion secretion. Our method can be easily adopted to study the biology of the ASDN and to investigate phenotypic differences between wild-type and transgenic mouse models.

Long-standing experimental evidence suggests that epithelial cells in the renal tubule are able to sense osmotic and pressure gradients caused by alterations in ultrafiltrate flow by elevating intracellular Ca2+concentration. These responses are viewed as critical regulators of a variety of processes ranging from transport of water and solutes to cellular growth and differentiation. A loss in the ability to sense mechanical stimuli has been implicated in numerous pathologies associated with systemic imbalance of electrolytes and to the development of polycystic kidney disease. The molecular mechanisms conferring mechanosensitive properties to epithelial tubular cells involve activation of transient receptor potential (TRP) channels, such as TRPV4, allowing direct Ca2+influx to increase intracellular Ca2+concentration. In this review, we critically analyze the current evidence about signaling determinants of TRPV4 activation by luminal flow in the distal nephron and discuss how dysfunction of this mechanism contributes to the progression of polycystic kidney disease. We also review the physiological relevance of TRPV4-based mechanosensitivity in controlling flow-dependent K+secretion in the distal renal tubule.

High-conductance (BK or maxi) K+channels were observed in cell-attached patches of the apical membrane of the isolated split-open rat connecting tubule (CNT). These channels were quite rare in cells identified visually as principal cells (PCs; 5/162 patches) but common in intercalated cells (ICs; 24/26 patches). The BK-expressing intercalated cells in the CNT and cortical collecting duct (CCD) were characterized by a low membrane potential (−36 mV) under short-circuit conditions, measured from the reversal potential of the channel currents with similar K+concentrations on both sides of the membrane. Under whole-cell clamp conditions with low intracellular Ca2+, ICs had a very low K+conductance. When cell Ca2+was increased to 200 nM, a voltage-dependent, tetraethylammonium (TEA)-sensitive outward conductance was activated with a limiting value of 90 and 140 nS/cell in the CNT and CCD, respectively. Feeding animals a high-K diet for 1 wk did not increase these currents. TEA-sensitive currents were much smaller in PCs and usually below detection limits. To examine the possibility that the ICs participate in transepithelial K+secretion, we measured Na/K pump activity as a ouabain-sensitive current. Although these currents were easily observed in PCs, averaging 79 ± 14 and 250 ± 50 pA/cell in the CCD and CNT, respectively, they were below the level of detection in the ICs. We conclude that ICs have BK channel densities that are sufficient to support renal secretion of K+if cell Ca2+is elevated. However. a pathway for K+entry into these cells has not been identified.

Thiazide and loop diuretics induce renal K+secretion, often leading to renal K+wasting and hypokalemia. This phenomenon has been proposed to reflect an increase in delivery to and reabsorption of Na+by the distal nephron, with a resultant increase in the driving force for passive K+efflux across the apical membrane. Recent studies suggest that cellular mechanisms that lead to enhanced rates of Na+reabsorption as well as K+secretion in response to increases tubular flow rates are more complex. Increases in tubular flow rates directly enhance the activity of apical membrane Na+channels and indirectly activate a class of K+channels, referred to as maxi-K, that are functionally inactive under low flow states. This review addresses the role of biomechanical forces, generated by variations in urinary flow rate and tubular fluid volume, in the regulation of transepithelial Na+and K+transport in the distal nephron. The question of why the distal nephron has evolved to include a component of flow-dependent K+secretion is also addressed.

Large, Ca2+-activated K+channels (BK), comprised of α- and β-subunits, mediate K+secretion during high flow rates in distal nephron segments. Because the BK-β1 subunit enhances Ca2+sensitivity of BK in a variety of cells, we determined its role in flow-induced K+secretion and its localization in the mammalian nephron. To determine the role of BK-β1 in the kaliuretic response to volume expansion, the rate of K+excretion (UKV) vs. varied urinary flow rates were determined in wild-type and BK-β1 knockout mice (BK-β1−/−). When flow rate was varied by volume expansion (2 ml·h−1·25 g body wt−1) for 30 to 60 min in wild-type mice, we found that the UKV increased significantly with increasing urine flow rates ( r2= 0.50, P < 0.00001, n = 31), as demonstrated previously in distal nephron of rats and rabbits. However, in BK-β1−/−mice, UKV did not vary with changing flow rates ( r2= 0.15, P = 0.08, n = 20). Using immunohistochemical techniques, we found that BK-β1 was strongly expressed in the apical membrane of the murine distal nephron and that 98% of BK-β1 protein detected by histochemistry colocalized with NCX, a marker of connecting tubules (CNT). Both BK-β1 and NCX colocalized with BK-α in separate experiments. Furthermore, we confirmed BK-β1 protein expression in the apical membrane of connecting tubules in rabbits. BK-β1 RNA from rabbit CNT was sequenced and was identical to previously published rabbit muscle sequences. These data show that the BK-β1 accessory subunit is present in the CNT segment of the mammalian distal nephron and has a significant role in the kaliuretic response to increased urinary flow induced by volume expansion.

Potassium secretory flux ( JK) by the distal nephron is regulated by systemic and luminal factors. In the present investigation, JKwas measured with a double-barreled K+electrode during paired microperfusion of superficial segments of the rat distal nephron. We used control solutions (100 mM NaCl, pH 7.0) and experimental solutions in which Cl−had been replaced with a less permeant anion and/or pH had been increased to 8.0. JKincreased when Cl−was replaced by either acetate (∼37%), sulfate (∼32%), or bicarbonate (∼62%), and also when the pH of the control perfusate was increased (∼26%). The majority (80%) of acetate-stimulated JKwas Ba2+sensitive, but furosemide (1 mM) further reduced secretion (∼10% of total), suggesting that K+-Cl−cotransport was operative. Progressive reduction in luminal Cl−concentration from 100 to 20 to 2 mM caused increments in JKthat were abolished by inhibitors of K+-Cl−cortransport, i.e., furosemide and [(dihydroindenyl)oxy]alkanoic acid. Increasing the pH of the luminal perfusion fluid also increased JKeven in the presence of Ba2+, suggesting that this effect cannot be accounted for only by K+channel modulation of K+secretion in the distal nephron of the rat. Collectively, these data suggest a role for K+-Cl−cotransport in distal nephron K+secretion.

The inwardly rectifying, ATP-sensitive K+ channel (ROMK) was localized by in situ hybridization in the rat kidney. Tissue in situ hybridization revealed that transcripts encoding the ROMK channel were expressed predominantly in cortical and outer medullary nephron segments. The localization of ROMK mRNA to specific nephron segments was assessed by hybridization of isolated nephron segments with an ROMK-specific probe (single segment in situ hybridization). ROMK mRNA was present in cortical and medullary thick ascending limb, distal tubule, and cortical and outer medullary collecting ducts, but not in proximal tubule. A weak hybridization was observed with inner medullary collecting ducts. To confirm these results, serial cryosections were alternatively stained by hybridization histochemistry for ROMK mRNA or by immunocytochemistry using antibodies specific for S1, S2, or S3 proximal tubular segments. Tubular cells that displayed immunoreactivity with the proximal tubular segment-specific antibodies showed little, if any, ROMK message. In addition, using an in situ hybridization and immunocytochemistry double-labeling technique, ROMK transcripts and vitamin D-dependent calcium-binding protein were shown to colocalize to the distal tubule (distal convoluted tubule and connecting tubule). The overall nephron localization of ROMK mRNA shown in these studies is consistent with the possibility that this novel channel may represent the low-conductance ATP-sensitive K+ channel that has been identified in apical membranes of thick limb and collecting duct segments and is believed to participate in K+ secretion.

the aldosterone-sensitive distal nephron, comprising the distal convoluted tubule (DCT), connecting tubule (CNT), cortical collecting duct (CCD), and outer medullary collecting duct, plays a major role in controlling Na+ and K+ homeostasis. A key mechanism, characterized mostly in the CCD, involves

this issue of the American Journal of Physiology : Renal Physiology contains two important papers describing the immunolocalization of transporters in the distal tubules of the mouse ([3][1], [6][2]). There are many reasons for the special interest in the organization of this region of the mouse

The effect of acute respiratory alkalosis (ARA) on distal nephron H+ secretion was evaluated by measuring urine-to-blood (U-B) Pco2 in dogs with highly alkaline urine (urine pH greater than 7.8). ARA led to a significant decrease in U-B Pco2 and in urine HCO3 concentration; urine pH, however, increased significantly, indicating that the decrease in urine Pco2 was of greater magnitude than the decrease in urine HCO3 concentration. For any given urine HCO3 concentration urine Pco2 was lower (i.e., urine pH was higher) in ARA than in controls. Administration of tris(hydroxymethyl)aminomethane (Tris) during ARA resulted in a significant increase in U-B Pco2 to control values. In animals with moderately alkaline urine (urine pH 6.4--7.4) and high urine PO4 concentration, ARA resulted in a significant decrease in UB-Pco2 and urine PO4 concentrations. Neutral PO4 infusion in these dogs resulted in an increase in urine PO4 concentration and U-B Pco2 to control levels. These data demonstrate that ARA results in a significant decrease in U-B Pco2 that is not solely attributable to changes in urine HCO3 concentration. The observation that Tris and PO4 infusion during ARA raises U-B Pco2 to control levels suggests that the ability to secrete H+ is intact.

In order to examine the role of dietary intake of phosphate in regulating renal phosphate transport, clearance and micropuncture studies were performed in 25 dogs with various duration of phosphate deprivation induced by low phosphate diet and aluminum hydroxide gel. In phosphate deprivation of 17-41 days duration, the phosphaturic response to extracellular volume expansion (ECVE) was blunted in the intact group and was virtually abolished in the acutely thyroparathyroidectomized (TPTX) group. With longer phosphate deprivation of 53-110 days, no phosphaturia occurred after ECVE or administration of parathyroid hormone (PTH), even with intact parathyroids. These alterations in phosphaturic responses did not correlate with the plasma phosphate but rather with the duration of phosphate deprivation. Fractional proximal tubule phosphate reabsorption was enhanced in phosphate deprivation and the proximal tubule fluid-to-ultrafilterable phosphate ratio appeared to be a good index for the degree of phosphate deprivation. Acute infusion of phosphate to raise plasma phosphate slightly above normal did not completely restore the responsiveness to ECVE. It is concluded that the adaptive response to phosphate deprivation occurs in both proximal and distal nephron segments and that factors other than plasma phosphate are primarily responsible for such an adaptation.