Medullary Tip Research Articles

Mammalian urine concentration: a review of renal medullary architecture and membrane transporters.

Mammalian kidneys play an essential role in balancing internal water and salt concentrations. When water needs to be conserved, the renal medulla produces concentrated urine. Central to this process of urine concentration is an osmotic gradient that increases from the corticomedullary boundary to the inner medullary tip. How this gradient is generated and maintained has been the subject of study since the 1940s. While it is generally accepted that the outer medulla contributes to the gradient by means of an active process involving countercurrent multiplication, the source of the gradient in the inner medulla is unclear. The last two decades have witnessed advances in our understanding of the urine-concentrating mechanism. Details of medullary architecture and permeability properties of the tubules and vessels suggest that the functional and anatomic relationships of these structures may contribute to the osmotic gradient necessary to concentrate urine. Additionally, we are learning more about the membrane transporters involved and their regulatory mechanisms. The role of medullary architecture and membrane transporters in the mammalian urine-concentrating mechanism are the focus of this review.

NSAIDs Alter Phosphorylated Forms of AQP2 in the Inner Medullary Tip.

Vasopressin increases urine concentration through activation of aquaporin-2 (AQP2) in the collecting duct. Nonsteroidal anti-inflammatory drugs (NSAIDs) block prostaglandin E2 synthesis, and may suppress AQP2 producing a urine concentrating defect. There are four serines in AQP2 that are phosphorylated by vasopressin. To determine if chronic use of NSAIDs changes AQP2’s phosphorylation at any of these residues, the effects of a non-selective NSAID, ibuprofen, and a COX-2-selective NSAID, meloxicam, were investigated. Daily ibuprofen or meloxicam increased the urine output and decreased the urine osmolality significantly by days 7 through 14. Concomitantly, meloxicam significantly reduced total AQP2 protein abundance in inner medulla (IM) tip to 64% of control and base to 63%, respectively. Ibuprofen significantly decreased total AQP2 in IM tip to 70% of control, with no change in base. Meloxicam significantly increased the ratios of p256-AQP2 and p261-AQP2 to total AQP2 in IM tip (to 44% and 40%, respectively). Ibuprofen increased the ratio of p256-AQP2 to total AQP2 in IM tip but did not affect p261-AQP2/total AQP2 in tip or base. Both ibuprofen and meloxicam increased p264-AQP2 and p269-AQP2 ratios in both tip and base. Ibuprofen increased UT-A1 levels in IM tip, but not in base. We conclude that NSAIDs reduce AQP2 abundance, contributing to decreased urine concentrating ability. They also increase some phosphorylated forms of AQP2. These changes may partially compensate for the decrease in AQP2 abundance, thereby lessening the decrease in urine osmolality.

Water-pumping turbulent flow in thin segments of the loop of Henle concentrates urine

In the mammalian kidney, urine is concentrated by passing through the thin descending limb (TDL) of Henle's loop, correlated with an osmotic gradient in the interstitial fluid that is increased from the outer medulla (OM) to the tip of the in- ner medulla (IM). The water-pumping turbulence hypothe- sis states that both the urinary concentrat- ing and the osmotic gradient of the renal medulla result from turbulent urine flow in the TDL. In turbulent flow, molecules move crosswise as well as along the tubule so H 2 O molecules are driven out through aquaporins; this concentrates urine. Since the TDL has fewer aquaporins in the IM than in the OM, the IM interstitium re- ceives less water; this can establish an osmotic gradient. Turbulence in thin seg- ments results from a greater Reynolds' number (RN), the measure of tendency for turbulence, calculated based on velocity, density, and viscosity of urinary flow and tubule diameter. RN (and the tendency for turbulence) of urinary flow in the TDL is approximately 2 times the RN in a thick segment. It makes the flow a water-pump and results in urinary concentration and the osmotic gradient of the renal medulla. INTRODUCTION The mammalian kidney continuously changes water excretion and urinary concentration depending on different conditions in order to maintain a nearly constant blood plasma osmolality. For most mammals that do not have free access to water, the ability to change wa- ter excretion is essential for survival 1 . In order to change urinary concentration, nephrons have several mechanisms, the best known of which is urinary concen- tration through the generation of a pro- gressive osmotic gradient extending from the cortico-medullary boundary to the inner medullary tip 2 and a hyperos-

Advances in Understanding the Urine-Concentrating Mechanism

The renal medulla produces concentrated urine through the generation of an osmotic gradient that progressively increases from the cortico-medullary boundary to the inner medullary tip. In the outer medulla, the osmolality gradient arises principally from vigorous active transport of NaCl, without accompanying water, from the thick ascending limbs of short- and long-looped nephrons. In the inner medulla, the source of the osmotic gradient has not been identified. Recently, there have been important advances in our understanding of key components of the urine-concentrating mechanism, including (a) better understanding of the regulation of water, urea, and sodium transport proteins; (b) better resolution of the anatomical relationships in the medulla; and (c) improvements in mathematical modeling of the urine-concentrating mechanism. Continued experimental investigation of signaling pathways regulating transepithelial transport, both in normal animals and in knockout mice, and incorporation of the resulting information into mathematical simulations may help to more fully elucidate the mechanism for concentrating urine in the inner medulla.

The Physiology of Urinary Concentration: An Update

The renal medulla produces concentrated urine through the generation of an osmotic gradient extending from the cortico-medullary boundary to the inner medullary tip. This gradient is generated in the outer medulla by the countercurrent multiplication of a comparatively small transepithelial difference in osmotic pressure. This small difference, called a single effect, arises from active NaCl reabsorption from thick ascending limbs, which dilutes ascending limb flow relative to flow in vessels and other tubules. In the inner medulla, the gradient may also be generated by the countercurrent multiplication of a single effect, but the single effect has not been definitively identified. There have been important recent advances in our understanding of key components of the urine concentrating mechanism. In particular, the identification and localization of key transport proteins for water, urea, and sodium, the elucidation of the role and regulation of osmoprotective osmolytes, better resolution of the anatomical relationships in the medulla, and improvements in mathematic modeling of the urine concentrating mechanism. Continued experimental investigation of transepithelial transport and its regulation, both in normal animals and in knock-out mice, and incorporation of the resulting information into mathematic simulations, may help to more fully elucidate the inner medullary urine concentrating mechanism.

Candesartan augments compensatory changes in medullary transport proteins in the diabetic rat kidney

Volume depletion due to persistent glucosuria-induced osmotic diuresis is a significant problem in uncontrolled diabetes mellitus (DM). Angiotensin II receptor blockers (ARBs), such as candesartan, slow the progression of chronic kidney disease in patients with DM. However, mice with genetic knockout of components of the renin-angiotensin system have urine concentrating defects, suggesting that ARBs may exacerbate the volume depletion. Therefore, the effect of candesartan on UT-A1, UT-A3, NKCC2, and aquaporin-2 (AQP2) protein abundances was determined in control and 3-wk DM rats. Aldosterone levels in control rats (0.36 +/- 0.06 nM) and candesartan-treated rats (0.34 +/- 0.14 nM) were the same. DM rats had higher aldosterone levels (1.48 +/- 0.37 nM) that were decreased by candesartan (0.97 +/- 0.26 nM). Western analysis showed that UT-A1 expression was increased in DM rats compared with controls in inner medullary (IM) tip (158 +/- 13%) and base (120 +/- 25%). UT-A3 abundance was increased in IM tip (123 +/- 11%) and base (146 +/- 17%) of DM rats vs. controls. UT-A3 was unchanged in candesartan-treated control rats. In candesartan-treated DM rats, UT-A3 increased in IM tip (160 +/- 14%) and base (210 +/- 19%). Candesartan-treated DM rats had slightly higher AQP2 in IM (46%, P < 0.05) vs. control rats. NKCC2/BSC1 was increased 145 +/- 10% in outer medulla of DM vs. control rats. We conclude that candesartan augments compensatory changes in medullary transport proteins, reducing the losses of solute and water during uncontrolled DM. These changes may represent a previously unrecognized beneficial effect of type 1 ARBs in DM.

Transport proteins in the inner medullas of diabetic kidneys are further increased by candesartan.

Dilute urine is a problem in uncontrolled diabetes mellitus (DM). Angiotensin II (AngII) receptor blockers ie. candesartan slow chronic kidney disease in DM patients. We investigated UT‐A1, UT‐A3, NKCC2, and AQP2 in control +/− candesartan rats and 3‐week DM +/− candesartan rats. Aldosterone levels in control rats (0.36 ± 0.06 nM) and candesartan‐treated rats (0.34±0.14 nM) were the same. DM rats had higher aldosterone levels (1.48±0.37 nM) that were decreased by candesartan (0.97±0.26 nM). Western blot analysis showed that in inner medullary (IM) tip and base, UT‐A1 expression was increased in DM rats compared to controls (Tip: 158±13%, Base: 120±25). UT‐A3 abundance was increased in both IM tip (123±11%) and base (146±17%) of DM rats vs controls (100%). UT‐A3 was unchanged in candesartan‐treated control rats. In candesartan treated DM rats UT‐A3 increased (IM tip: 160±14%; base 210±19%). Candesartan treated DM rats had slightly higher AQP2 in IM (46%, p&lt;0.05) vs control rats. NKCC2 was increased 45±10% in OM of DM vs control rats. While candesartan did not alter NKCC2 in controls, NKCC2 was increased a further 41% by candesartan treatment in DM rats. We conclude that candesartan augments compensatory changes in medullary transport proteins, reducing the losses of solute and water during uncontrolled DM. These changes may represent a previously unrecognized beneficial effect of AngII receptor blockers in diabetes.

Physiology of Renal Medullary Tip Hyperattenuation at Unenhanced CT: Urinary Specific Gravity and the NaCl Concentration Gradient

To retrospectively investigate the physiology of renal medullary tip hyperattenuation at unenhanced computed tomography (CT). This retrospective single-institution study was IRB approved and HIPAA compliant. Informed consent was waived. One hundred consecutive patients (53 women, mean age, 52 years; 47 men, mean age, 48 years; P = .39) without and 34 (11 women, mean age, 49 years; 23 men, mean age, 45 years; P = .54) with unilateral ureteral obstruction underwent contemporaneous urinalysis and unenhanced CT. At CT, bladder urine attenuation was measured and two readers recorded the presence of renal medullary tip hyperattenuation. For obstructed kidneys (n = 34), renal pelvic urine attenuation was also recorded. The presence of medullary tip hyperattenuation was correlated with urinary specific gravity. To investigate the physiologic basis of medullary tip hyperattenuation, attenuations for NaCl and urea phantoms (range, 0-2000 mosm/kg) were recorded and correlated to solute concentrations by using linear regression. Patients with renal medullary tip hyperattenuation seen at CT had higher mean urinary specific gravity (1.023 and 1.022 for readers 1 and 2, respectively) than those without (1.015 and 1.016, respectively, both P < .05). The specific gravity correlated with higher urine attenuation (r = 0.40, P < .001). For the 34 patients with unilateral urinary obstruction, medullary tip hyperattenuation was less commonly seen in obstructed (two kidneys each for both readers) than nonobstructed (11 and 15 kidneys, respectively, both P < .005) kidneys and mean urine attenuation was lower in the obstructed renal pelvis (7.4 HU) than in the bladder (11.4 HU) (P < .005). Phantoms showed a 3.6-HU increase per 100-mosm/kg increase in NaCl concentration (r = 0.99, P < .001) but no change in attenuation with different urea concentrations. Renal medullary tip hyperattenuation at unenhanced CT reflects increased urinary specific gravity, likely related to high medullary tip NaCl concentrations.

Forskolin stimulates phosphorylation and membrane accumulation of UT-A3

UT-A1 is regulated by vasopressin and is localized to the apical membrane and intracellular compartment of inner medullary collecting duct (IMCD) cells. UT-A3 is also expressed in the IMCD and is regulated by forskolin in heterologous systems. The goal of the present study is to investigate mechanisms by which vasopressin regulates UT-A3 in rat IMCD. In fresh suspensions of rat IMCD, forskolin increases the phosphorylation of UT-A3, similar to UT-A1. Biotinylation studies indicate that UT-A3 is located in the plasma membrane. Forskolin treatment increases the abundance of UT-A3 in the plasma membrane similar to UT-A1. However, these two transporters do not form a complex through a protein-protein interaction, suggesting that transporter function is unique to each protein. While immunohistochemistry localized UT-A3 to the basal and lateral membranes, a majority of the staining was cytosolic. Immunohistochemistry of vasopressin-treated rat kidney sections also localized UT-A3 primarily to the cytosol with basal and lateral membrane staining but also showed some apical membrane staining in some IMCD cells. This suggests that under normal conditions, UT-A3 functions as the basolateral transporter but in a high cAMP environment, the transporter may move from the cytosol to all plasma membranes to increase urea flux in the IMCD. In summary, this study confirms that UT-A3 is located in the inner medullary tip where it is expressed in the basolateral membrane, shows that UT-A3 is a phosphoprotein in rat IMCD that can be trafficked to the plasma membrane independent of UT-A1, and suggests that vasopressin may induce UT-A3 expression in the apical plasma membrane of IMCD.

Adrenalectomy Blocks the Compensatory Increases in UT-A1 and AQP2 in Diabetic Rat Kidney

In normal rats we showed that glucocorticoids participate in the downregulation of UT-A1 protein abundance in the inner medullary tip and in lowering of basal and vasopressin-stimulated facilitated urea permeability in terminal IMCDs. To examine the relevance of this response to a rat model of human disease, we studied rats with uncontrolled diabetes mellitus (DM) induced by streptozotocin (STZ), since these rats have increased corticosterone production and urea excretion. We found that at 3 days of DM, UT-A1 protein abundance is downregulated in the inner medullary tip compared to pair-fed control rats, while DM for more than 7 days caused an increase in UT-A1. To test whether adrenal steroids could be a mechanism contributing to the latter increase, we studied adrenalectomized rats (ADX), ADX rats given STZ to induce diabetes (ADX + STZ), and ADX + STZ rats receiving exogenous aldosterone or dexamethasone. In contrast to control rats, UT-A1 protein abundance was not increased by prolonged DM in the ADX rats. Aquaporin 2 (AQP2) was not increased in the inner medullas of 10-day DM rats either. However, UT-A1 protein abundance was significantly reduced in the inner medullary tips from both diabetic aldosterone-treated (40 +/- 2%) and dexamethasone-treated (43 +/- 2%) ADX rats compared to diabetic ADX rats without steroid replacement. AQP2 was unaffected by steroid hormone treatments. Thus, both mineralocorticoids and glucocorticoids downregulate UT-A1 protein abundance in rats with uncontrolled diabetes mellitus for 10 days. These results suggest that: 1) the increase in UT-A1 observed in DM is dependent upon having adrenal steroids present; and 2) adrenal steroids are not sufficient to enable the compensatory rise in UT-A1 to a steroid-deficient diabetic animal.

Urea transporter UT-A1 and aquaporin-2 proteins decrease in response to angiotensin II or norepinephrine-induced acute hypertension

The kidney responds to high levels of ANG II, as may occur during malignant hypertension, by increasing sodium and water excretion. To study whether kidney medullary transporters contribute to this response, rats were made hypertensive using ANG II. Within 3 days of being given ANG II, systolic blood pressure (BP) was increased (200 mmHg), vs control (130 mmHg), and remained high through day 14. Kidney inner medullary (IM) tip and base and outer medulla were analyzed for transporter protein abundance. There were significant decreases in UT-A1 urea transporter, aquaporin-2 (AQP2) water channel, and NKCC2/BSC1 Na(+)-K(+)-2Cl(-) cotransporter. To determine whether the decreases were a response to hypertension, ANG II, or an ANG II-induced increase in aldosterone, rats were given 1) norepinephrine (to increase BP) and 2) ANG II plus spironolactone (to block the mineralocorticoid receptor). Norepinephrine (7 days) increased BP, urine volume, sodium excretion, and decreased urine osmolality and UT-A1, AQP2, and NKCC2/BSC1 abundances, similar to ANG II. ANG II alone or with spironolactone yielded similar increases in BP, urine volume, and urine osmolality, and decreases in UT-A1 and AQP2 proteins in the IM tip. Plasma vasopressin was unaffected by treatment. Water diuresis did not change UT-A1 but decreased AQP2 and NKCC2/BSC1 abundances. We conclude that decreases in UT-A1, AQP2, and NKCC2/BSC1 proteins may contribute to the diuresis and natriuresis that occur following ANG II or norepinephrine-induced acute hypertension and do not appear to involve ANG II stimulation of aldosterone or thirst.

Aldosterone Decreases UT-A1 Urea Transporter Expression via the Mineralocorticoid Receptor

Adrenalectomy in rats is associated with urinary concentrating and diluting defects. This study tested the effect of adrenal steroids on the UT-A1 urea transporter because it is involved in the urine-concentrating mechanism. Rats were adrenalectomized and given normal saline for 14 d, after which they received (1) vehicle, (2) aldosterone, or (3) spironolactone plus aldosterone. Adrenalectomy alone significantly increased UT-A1 protein in the inner medullary tip after 7 d, whereas aldosterone repletion reversed the effect. Spironolactone blocked the aldosterone-induced decrease in UT-A1, indicating that aldosterone was working via the mineralocorticoid receptor. For verifying that glucocorticoids downregulate UT-A1 protein through a different receptor, three groups of adrenalectomized rats were prepared: (1) vehicle, (2) adrenalectomy plus dexamethasone, and (3) adrenalectomy plus dexamethasone and spironolactone. Dexamethasone significantly reversed UT-A1 protein abundance increase in the inner medullary tip of adrenalectomized rats. When spironolactone was given with dexamethasone, it did not affect the dexamethasone-induced decrease in UT-A1. There was no significant change in serum vasopressin level, aquaporin 2, or Na(+)-K(+)-2Cl(-) co-transporter NKCC2/BSC1 protein abundances or UT-A1 mRNA abundance in any of the groups. In conclusion, either mineralocorticoids or glucocorticoids can downregulate UT-A1 protein. The decrease in UT-A1 does not require both steroid hormones, and each works through a different receptor.

Changes in renal medullary transport proteins during uncontrolled diabetes mellitus in rats.

We tested whether the abundance of transport proteins involved in the urinary concentrating mechanism was altered in rats with uncontrolled diabetes mellitus (DM). Rats were injected with streptozotocin and killed 5, 10, 14, or 20 days later. Blood glucose in DM rats was 300-450 mg/dl (control: 70-130 mg/dl). Urine volume increased in DM rats from 41 +/- 7 ml/100 g body wt (BW) at 5 days to 69 +/- 3 ml/100 g BW at 20 days (control: 9 +/- 1). Urine osmolality of DM rats decreased at 5 days DM and remained low at 20 days. UT-A1 urea transporter protein in the inner medullary (IM) tip was 55% of control in 5-day DM rats but increased to 170, 220, and 280% at 10, 14, and 20 days DM, respectively, due to an increase in the 117-kDa glycoprotein form. UT-A1 in the IM base was increased to 325% of control at 5 days DM with no further increase at 20 days. Aquaporin-2 (AQP2) increased to 290% in the IM base at 5 days DM and 150% in the IM tip at 10 days; both showed no further increase at 20 days. NKCC2/BSC1 increased to 240% in outer medulla at 20 days DM, but not at 5 or 10 days. UT-B and ROMK were unchanged at any time point. The increases in UT-A1, AQP2, and NKCC2/BSC1 proteins during uncontrolled DM would tend to limit the loss of fluid and solute during uncontrolled diabetes.

Glucocorticoids mediate a decrease in AVP-regulated urea transporter in diabetic rat inner medulla.

Providing glucocorticoids to adrenalectomized (Adx) rats results in downregulation of the vasopressin (AVP)-regulated urea transporter (VRUT) in the renal inner medullary (IM) tip. To examine the physiological relevance of this response, we studied rats with uncontrolled diabetes mellitus induced by streptozotocin (STZ), since these rats have increased corticosterone production and urea excretion. We measured VRUT protein in extracts from the IM tip or base of pair-fed control and diabetic rats by Western analysis using an antibody to rat VRUT. In the IM tip, VRUT was significantly reduced by 39% in diabetic compared with control rats. In the IM base, there was no significant difference between diabetic and control rats. To determine whether the decrease in VRUT in the IM tip was mediated by glucocorticoids, the experiment was repeated using the following three groups of rats: 1) Adx alone, 2) Adx + STZ, and 3) Adx + STZ + replacement with a physiological dose of glucocorticoid. There was no significant difference in VRUT between Adx and Adx + STZ rats. However, VRUT was significantly reduced by 32% in the IM tip of glucocorticoid-treated Adx + STZ rats compared with control Adx + STZ rats. We conclude that glucocorticoids regulate the abundance of VRUT protein independently of insulin in diabetic rats.

Glucocorticoids downregulate the vasopressin-regulated urea transporter in rat terminal inner medullary collecting ducts.

This study tested whether glucocorticoids regulate tubular urea transport. Urea permeability was measured in perfused inner medullary collecting duct (IMCD) subsegments from rats that underwent adrenalectomy, adrenalectomy plus replacement with a physiologic dose of glucocorticoid (dexamethasone), or sham operation. Compared with sham rats, basal urea permeability in terminal IMCD was significantly increased in adrenalectomized rats and reduced in dexamethasone-treated rats. Vasopressin significantly increased urea permeability in all three groups. In contrast, there was no difference in basal or vasopressin-stimulated urea permeability in initial IMCD between the three groups. Next, membrane and vesicle fraction proteins were isolated from inner medullary tip or base and Western analysis was performed by use of an antibody to the rat vasopressin-regulated urea transporter. Vasopressin-regulated urea transporter protein was significantly increased in both membrane and vesicle fractions from the inner medullary tip of adrenalectomized rats. There was no change in vasopressin-regulated urea transporter protein in the inner medullary base, and Northern analysis showed no change in urea transporter mRNA abundance in either inner medullary region. It was concluded that glucocorticoids can downregulate function and expression of the vasopressin-regulated urea transporter in rat terminal IMCD.

Distribution of aquaporin-4 water channel expression within rat kidney

The aquaporins are a family of transmembrane proteins that function as molecular water channels. Recently, a mercurial-insensitive water channel [MIWC or aquaporin-4 (AQP4)] has been cloned, and its mRNA was found to be expressed strongly in kidney inner medulla and several nonrenal tissues. We prepared affinity-purified polyclonal antipeptide antibodies to AQP4 to define the regional distribution and cellular location of this water channel within the kidney. Immunoblotting of membrane fractions from different regions of the kidney revealed strongest expression in the base of the renal inner medulla, with detectable levels also in the inner medullary tip, but little or no expression in the outer medulla or cortex. Immunocytochemistry (light microscopy) revealed renal AQP4 labeling exclusively in the collecting duct principal cells, chiefly in the proximal two-thirds of the inner medullary collecting duct (IMCD). Little or no expression was seen in the outer medullary and cortical collecting ducts. Immunoelectron microscopy demonstrated AQP4 labeling of the basolateral membrane of IMCD cells, with relatively little labeling of intracellular vesicles. Differential centrifugation of inner medullary homogenates also revealed a lack of distribution to the vesicle-enriched fraction, which contains the vasopressin-regulated water channel, aquaporin-2. In contrast to aquaporin-2 and aquaporin-3, water restriction of rats did not increase the level of AQP4 expression. These results suggest a possible role for AQP4 in the basolateral exit of water from the IMCD.

Comparison of the renal medullary vascular systems of dog and chinchilla

Renal vascular structures of dogs and chinchillas were investigated, using a low viscosity silicone rubber arterial injection technique. Cortical and medullary thicknesses were measured and compared to measurements reported by Sperber. Cortical and juxtamedullary structural differences were found in the dog and chinchilla. The juxtamedullary glomeruli had larger diameters, longer afferent and efferent arterioles, and much larger efferent arteriole lumen diameters compared to cortical glomeruli of the same kidney. The vasa recta bundles of dogs are of larger over-all diameter but of approximately the same length and spacing as in the chinchilla. A model for the medullary circulation has been proposed. This model represents three major flow routes to convoluted tubule capillaries, frizzled zone capillaries, and medullary tip capillaries in series with the juxtamedullary glomeruli. The probable contribution of countercurrent exchange of diffusible solutes, occurring between the medullary venous drainage and inflowing blood and its role in the conservation of medullary solutes is discussed.