Mechanisms to concentrate the urine: an opinion

PURPOSE OF REVIEW: Our goal is to suggest how the renal concentrating mechanism is regulated in vivo. RECENT FINDINGS: The majority of descending thin limbs of the loop of Henle lack aquaporin-1 water channels, and loops of Henle in the inner medulla lack urea transporters. SUMMARY: Lack of water permeability in the descending thin limbs of the loop of Henle offers several advantages. First, since much less water is added to the outer medullary interstitial compartment, inhibitory control mechanisms on sodium and chloride reabsorption from the medullary thick ascending of loop of Henle initiated by water addition from the medullary collecting duct can be effective. Second, recycling of urea is efficient, as little urea will be washed out of the medulla. Third, delivery of a larger volume of filtrate to the medullary thick ascending limb of the loop of Henle permits both an appreciable reabsorption of sodium along with only a small fall in the luminal concentration of sodium in each of these liters. Hence there need be only a small lumen positive voltage in the medullary thick ascending limb of the loop of Henle. The absence of urea transporters in the loop of Henle in the inner medulla is required for a passive mechanism of sodium and chloride reabsorption in the inner medulla. Control of urea reabsorption from the medullary collecting duct is needed to prevent excessive oliguria in electrolyte-poor urine.     

Aquaporin-1 is not expressed in descending thin limbs of short-loop nephrons

In mammalian kidneys, aquaporin-1 is responsible for water reabsorption along the proximal tubule and is also thought to be involved in the concentration of urine that occurs in the medulla. It has been suggested, however, that aquaporin-1 is not expressed in the last part of the descending thin limbs of short loop nephrons in rats and mice, and its expression in this region in humans has not been studied. We examined the expression of aquaporin-1 and the urea transporter UT-A2 in serial sections of mouse nephrons in the inner stripe of the outer medulla using immunohistochemistry. In contrast to previous observations, we demonstrate a complete absence of aquaporin-1 along the entire length of descending thin limbs of 90% of short loop nephrons. Conversely, as expected, we identified aquaporin-1 in proximal tubules, descending thin limbs of long loop nephrons, and medullary descending vasa recta. We also observed this abrupt transition from aquaporin-1-positive proximal tubules to aquaporin-1-negative descending thin limbs of short loop nephrons in sections of human and rat kidneys. UT-A2 was restricted to the last 28% to 44% of the descending thin limbs of all short loop nephrons. Because the majority of nephrons are of the short loop variety, our findings suggest that the mechanisms of water transport in the descending thin limbs of short loop nephrons should be reevaluated. Likewise, the roles of aquaporin-1 and UT-A2 in the countercurrent multiplier and water conversation may need to be readdressed.     

Short and long loop nephrons

The explanation for the necessity to have both short and long loop nephrons for urinary concentration is unknown but may represent nature's resolution of conflicting ideal conditions for maximum urinary concentration. Ideally, one would like the thick ascending limb to extend throughout the entire medulla to the papillary tip and be supplied by a blood flow vigorous enough to provide oxygen and remove waste products as rapidly as needed. One would also like to have a progressively smaller volume of tissue to be concentrated toward the papillary tip to lessen the osmotic work required and a highly efficient vascular exchange system to sequester the medullary interstitial solute effectively. But the same efficiency of countercurrent exchange of oxygen causes the inner medulla to have a relatively low oxygen content. The presence of the thin loops of Henle in the inner medulla may represent a compromise between these conflicting ideals. The papilla tapers to a low mass, which allows a mechanism requiring only a modest energy supply to increase the tonicity of the interstitium enormously. The reduced work requirement obivates the need for thick ascending limbs to extend into the papilla where they would be highly vulnerable to anoxia. The outer medulla with its larger mass and thick ascending limbs supplied by a high blood flow can initiate the operation to reduce the volume of fluid and solute to be concentrated, and at the same time carry out other functions required of the filtration-reabsorption kidney.(ABSTRACT TRUNCATED AT 250 WORDS)     

The vulnerability of the thin descending limbs of Henle's loop in the isolated perfused rat kidney

In the isolated rat kidney perfused without erythrocytes, the medullary thick ascending limb shows extensive injury. Damage to the thin limbs of Henle's loop has been mentioned only briefly. Thin limbs were examined in the isolated perfused kidney under a variety of conditions that alter oxygenation and active transport in the medulla and are known to affect injury to the medullary thick ascending limb. The thin descending limbs of short loops were preserved in all experimental groups, but those of the long loop showed necrosis that was restricted to the proximal portion, where the epithelium is more complex. In oxygenated kidneys, necrosis involved 41% +/- 5% (mean +/- SE) of the medullary thick ascending limbs and 10% +/- 3% of the proximal portion of long loops of thin descending limbs. Under hypoxic conditions, necrosis involved 90% +/- 3% of the medullary thick ascending limbs and 70% +/- 5% of the proximal portion of long loops of thin descending limbs (P less than 0.0001 compared with oxygenated kidneys). Ouabain and absence of filtration completely prevented necrosis of both nephron segments. Thus, the proximal portions of long loops of thin descending limbs, in resemblance to medullary thick ascending limbs, are especially susceptible to transport-dependent hypoxic injury.     

Progesterone--specific binding sites in the kidney of the female baboon

The uptake and retention of a radiolabeled synthetic progestin, ORG 2058, was studied in the urinary tract of the female baboon. Four estrogen-primed baboons were injected intravenously with 2.5 micrograms./kg. body weight of 3H-ORG 2058. One animal, which served as a control, received an additional injection of 2.5 mg./kg. body weight of unlabeled progesterone. One hour after the injections, the animals were killed and the kidneys, ureters and urinary bladder were removed and processed for autoradiography. Localization of progestin was observed in the nuclei of the convoluted and straight segments of the distal tubule, the ascending thick limb of the loop of Henle and both cortical and medullary collecting tubules. Connective tissue cells were also labeled in the medulla and cortex of the kidney. An absence of silver grains was noted in the renal corpuscle, all segments of the proximal tubule and the thin loop of Henle. Concentration of the tritiated steroid was not observed in either the ureter or bladder or in any portions of the urinary tract of the control animal. This study suggests that progesterone has a direct effect via a progesterone specific receptor on the various target cells that sequestered the 3H-ORG 2058.     

Effect of epidermal growth factor on the developing rat renal papilla

BACKGROUND: Apoptosis plays an important role in the morphogenesis of the renal papilla. During kidney development, ATL is derived from the TAL in the inner medulla by apoptotic deletion of a fraction of TAL cells and the transformation of the remaining TAL cells. EGF is an important regulator of apoptosis in the kidney. HYPOTHESIS: Exogenously administered EGF in postnatal rat affects renal papilla growth with cell proliferation and apoptosis in the loop of Henle. METHODS: Rat pups received subcutaneous injections of EGF (0.3 microg/g body weight) or saline four times a day from after birth. Rats were sacrificed and the kidneys were preserved for immunohistochemistry on day 4 and day 7. The TAL was identified with antibody directed against Na-K-ATPase or BSC1, and type A intercalated cells were identified with antibody to anion exchanger 1 (AE1). Apoptosis was detected with TUNEL method, and cell proliferation with immunostaining for PCNA. RESULTS--PRIMARY AND SECONDARY: EGF treatment resulted in the following: (1) reduced kidney weight; (2) shortened length of renal papilla; (3) delayed transformation of the cuboidal epithelium into the squamous epithelium of the ATL; (4) delayed elimination of type A intercalated cells in the medullary collecting duct; (5) decreased both apoptotic index and PCNA-positive cells in the TAL and the collecting duct of the renal medulla. CONCLUSION: These findings suggest that exogenous EGF delays the development of loop of Henle in the renal papilla by reducing both apoptosis and cell proliferation.     

Regional membrane specialization in the thin limbs of Henle's loops as seen by freeze-fracture electron microscopy

Rat thin limbs of Henle were studied by freeze-fracture electron microscopy. Thin limb segments in both short- and long-looped nephrons were identified by previously developed ultrastructural criteria, continuity with known thick segments, and architectural relationships in the outer medulla. Intramembrane particle (IMP) density and the number of intramembrane fibrils comprising the zonula occludens were determined for each morphologically identifiable thin limb segment. The IMP density on the protoplasmic faces of both the luminal and abluminal membranes of the upper portion of the descending thin limb (DTL) of the long-looped nephron is quantitatively greater than in the short-looped thin limb, lower portion of the long-looped DTL, and in the ascending thin limb. The zonulae occludens in the long-looped upper DTL consists of a single fibril; the long-looped lower DTL contains 3.13 +/- 0.14 fibrils; the ascending thin limb contains 1.31 +/- 0.09 fibrils; and the short-looped DTL contains 3.75 +/- 0.19 fibrils. These studies further support the contention that there is anatomic heterogeneity among the thin limb segments. Because direct physiologic studies in the thin limbs are incomplete and conflicting, the need for correlative physiologic studies on anatomically characterized structures is indicated.     

Editorial review. Recent formulations of the urinary concentrating mechanism: a status report.

The status of the purely passive mode of solute concentration as of 1979 appears to be similar to that of the original countercurrent hypothesis 10 years ago. The passive mode concept has advanced our understanding of the concentrating process by qualitatively incorporating the permeability characteristics of tubule segments and the lack of an active transport process in the thin loop of Henle into a mechanism which has attractive economy and explanatory value. But in the final analysis some assumptions are not borne out by experimental findings (for example, the high urea concentration of fluid in the rat and hamster end-descending limb; the likelihood of net transepithelial addition of sodium chloride to the Psammomys descending limb; the removal of sodium chloride from the hamster ascending limb against an apparent electrochemical gradient under certain circumstances; and the osmotic lag between vasa recta blood and interstitium in the rat). Furthermore, when the known permeability and transport characteristics of the renal tubule are incorporated into a mathematic model of the passive operating mode, numerical simulations fail to establish a progressively hyperosmotic inner medulla. This does not rule out the applicability of the more general model (Eq. 1), particularly if evidence for some form of active transport in the inner medulla, heretofore lacking, is forthcoming.     

Intracellular distribution of carbonic anhydrase in the rat kidney

The rat kidney was studied by light and electron microscope after it was histochemically stained for carbonic anhydrase activity. Glomeruli and Bowman's capsule were inactive. Convoluted proximal tubules showed intense activity at the brush border and the basolateral membranes. Cytoplasmic activity also was found. Straight proximal tubules had considerable enzyme activity at basolateral membranes but only low activity at the brush border and in the cytoplasm. In nephrons with long loops, the descending thin limb contained cytoplasmic enzyme activity, whereas the ascending thin limb was inactive. Thin limbs of short loops showed a varying enzyme pattern. In the thick limb of Henle's loop, most enzyme activity was found at the luminal cell border. Distal convoluted tubules showed enzyme activity only at basal infolded membranes. In the late distal tubule, intercalated cells appear among the "ordinary" distal cells, and they contained abundant cytoplasmic enzyme. Many highly active intercalated cells were found also in the cortical and outer medullary segments of the collecting duct. The chief cells in these segments also showed some cytoplasmic enzyme activity. In the inner medullary segment of the collecting duct, enzyme activity disappeared gradually, and the tip of the papilla lacked activity. Acetazolamide (10 microM) completely abolished visible staining, whereas Cl 13850 (10 microM), an inactive acetazolamide analogue, did not interfere with the staining.     

Effect of loop diuretics on organic osmolytes and cell electrolytes in the renal outer medulla.

Electron microprobe analysis on freeze-dried cryosections was used to determine the effect of the loop diuretics torasemide and furosemide on intracellular electrolyte concentrations in individual cells of the outer and inner stripe of the outer medulla and on cell rubidium uptake, the latter a measure of basolateral Na-K-ATPase activity. In addition, the organic osmolytes glycerophosphorylcholine (GPC), betaine, inositol and sorbitol in cortex, outer medulla and inner medulla were measured using HPLC. Both loop diuretics significantly reduced sodium and chloride concentrations and rubidium uptake in thick ascending limb cells, but did not affect sodium concentration or rubidium uptake in the proximal straight tubule (PST) cells or in the light or dark cells of the outer medullary collecting duct (OMCD). Chloride concentrations in these cells (that is, PST cells, OMCD light and dark cells) were lowered by loop diuretics, albeit less than in thick ascending limb cells. Administration of both loop diuretics for only 20 minutes was sufficient to significantly depress tissue concentrations of GPC, betaine, and myo-inositol in the outer medulla and of GPC, betaine and sorbitol at the papillary tip. These results indicate that loop diuretics, presumably by blocking apical sodium entry, decrease thick ascending limb cellular sodium concentration and, as a consequence, reduce Na-K-ATPase activity as assessed by cell rubidium uptake. Although this has been shown previously in in vitro preparations, the present study confirms this for the first time in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)     

IL-18 is expressed in the intercalated cell of human kidney

We determined the cellular location of interleukin-18 (IL-18) and caspase-1 and the purinergic receptor P2X7, two proteins necessary for its activation and secretion. The mRNA and protein of IL-18 were detectable in normal human kidney by means of polymerase chain reaction (PCR), in situ hybridization, and Western blot. Immunohistochemistry located IL-18 to nephron segments containing calbinbin-D28k or aquaporin-2 that suggest location in the distal convoluted and the connecting tubule and to parts of the collecting duct. IL-18 was not detected in the thick ascending limb of Henle. Confocal microscopy showed that IL-18 was expressed in cells negative for calbindin-D28k and for aquaporin-2 but positive for the vacuolar H(+)-ATPase. This demonstrates that the intercalated cells produce IL-18. These segments were also positive for caspase-1 and P2X7 that are essential for IL-18 secretion. Our results show that IL-18 is constitutively expressed by intercalated cells of the late distal convoluted tubule, the connecting tubule, and the collecting duct of the healthy human kidney. Since IL-18 is an early component of the inflammatory cytokine cascade, its location suggests that renal intercalated cells may contribute to immediate immune response of the kidney.     

Mechanisms of Disease: the kidney-specific chloride channels ClCKA and ClCKB, the Barttin subunit, and their clinical relevance

Rodent ClC-K1 and ClC-K2, and their respective human orthologs ClCKA and ClCKB, are chloride channels specific to the kidney (and inner ear); Barttin is their functionally important subunit. ClC-K1 is predominantly localized to the thin ascending limb of the loop of Henle. ClC-K2 is expressed more broadly in the distal nephron; expression levels are highest along the thick ascending limb of the loop of Henle and distal convoluted tubule. Expression of ClC-K1 is upregulated by dehydration and downregulated by the diuretic furosemide, whereas expression of ClC-K2 is upregulated by furosemide and downregulated by high salt levels. ClCKA is important for maintenance of the corticomedullary osmotic gradient and the kidney's capacity to concentrate urine. If its ortholog, ClC-K1, is nonfunctional in mice, renal diabetes insipidus develops. ClCKB is a key determinant of tubular reabsorption of chloride and electrolytes along the distal tubule. A severe salt-losing tubulopathy (Bartter syndrome type III) develops if ClCKB is nonfunctional, whereas a common genetic variant of the CLCNKB gene that leads to increased activity of ClCKB results in salt-dependent hypertension. Disruption of the gene encoding Barttin, BSND, results in a 'double knockout' of the functions of both ClCKA and ClCKB, manifesting as Bartter syndrome type IV with sensorineural deafness and an especially severe salt-losing phenotype.     

Immunolocalization of protein kinase C isoenzymes alpha, beta1 and betaII in rat kidney.

Protein kinase C (PKC) significantly contributes to the control of renal function, but little is known about the renal function or localization of PKC isoenzymes. Therefore, the localization of PKC isoenzymes alpha, betaI, and betaII was studied in rat kidney. Immunoblot analysis identified immunoreactive bands corresponding to PKC a, betaI, and betaII in total cell extracts of both renal cortex and medulla. Immunohistochemistry using confocal laser scanning microscopy revealed immunostaining for PKC alpha within the glomerulus including podocytes and mesangial cells. PKC betaI was detected in mesangial cells, whereas anti-PKC betaII labeled neither podocytes nor mesangial cells. PKC betaII, however, was detected in cells within the mesangial area, which expressed MHC II, a marker for antigen-presenting cells. None of the three isoforms was detected in glomerular endothelial cells. A prominent immunostaining with anti-PKC alpha and betaI was localized to the brush border of S2 and S3 segments of proximal tubule, whereas S 1 segments were not stained. Along the loop of Henle, both PKC a and PKC betaI were found in the luminal membrane of cortical and medullary thick ascending limb. In addition, anti-PKC betaI labeled the luminal membrane of thin limbs. In the cortical collecting duct (CCD), immunofluorescence for PKC alpha was observed at the apical membrane of both peanut agglutinin (PNA)-negative cells and part of PNA-positive cells, whereas in the medullary collecting duct (MCD), PKC a was detected at the basolateral membrane. In comparison, PKC betaI was localized at the luminal membrane of PNA-positive cells only in CCD and at the luminal membrane of MCD. Unlike PKC a or betaI, there was (1) no detectable immunostaining with anti-PKC betaII in the proximal tubule, the loop of Henle, or the CCD and (2) a distinct staining for PKC betaII of interstitial cells in cortex and medulla (including MHC II-positive dendritic cells). Furthermore, PKC betaII was detected in the luminal membrane of MCD. In summary, a distinct and differential expression pattern for PKC alpha, betaI, and betaII was shown in rat kidney, which may contribute to a better understanding of the specific role of these isoenzymes in the control of renal function.     

High resolution localization of angiotensin II receptors in rat renal medulla.

The cellular localization of angiotensin II (Ang II) receptors in the inner stripe of the outer medulla of the rat kidney was investigated by using high resolution light and electron microscopic autoradiography. Fresh tissue blocks from the inner stripe of the outer medulla were incubated with 125I-[Sar1, Ile8] Ang II and prepared for microscopic autoradiography. At the light microscopic level, 125I-[Sar1, Ile8] Ang II was found to penetrate into the tissue and to bind specifically to sites outlining renal tubules and vasa recta bundles. Electron microscopic autoradiography revealed that silver grains were detected over interstitial cells located between the tubules and components of the vasa recta bundles, but no silver grains were detected overlying the cells of the thin descending or thick ascending limbs of the loop of Henle, the collecting ducts, the vasa recta, or other blood vessels. These interstitial cells contained abundant endoplasmic reticulum, microfilaments, occasional lipid droplets and extensive cytoplasmic processes which closely related to the basement membranes of the vasa recta and loops of Henle. The cells therefore closely resemble type 1 interstitial cells. Since Ang II binding sites are absent in the inner medulla, the cells labelled by this technique must be a subset of type 1 interstitial cells, distinct from the typical lipid-laden interstitial cells most abundant in the inner medulla. These findings demonstrate that type 1 interstitial cells are the primary sites for a high density of Ang II receptors located in the inner stripe of the outer medulla.     

Branched-chain amino acid aminotransferase along the rabbit and rat nephron

The activity of branched-chain amino acid aminotransferase (EC 2.6.1.42) is reported for four or five different segments of the rat and rabbit nephron as well as for patches from the papilla. In the rat the levels ranged 40-fold, from a high in the thick ascending limb of Henle to a low in the proximal convoluted tubule. The peak activity is far above that reported for most other parts of the body. Maximum activity was located also in the thick ascending limb in the rabbit, but the level was only one-third as high as in the rat. It is postulated that ammonia liberated by this amino transferase, in cooperation with glutamate dehydrogenase, could diffuse readily into the adjacent proximal straight tubule where all of the renal glutamine synthase and the highest level of alanine aminotransferase are located. Thus alanine and glutamine could be produced when the ammonia was not needed to neutralize excess acidity.     

Calbindin-D9k and parvalbumin are exclusively located along basolateral membranes in rat distal nephron.

There is strong evidence that vitamin D-dependent Ca(2+)-binding proteins, i.e., calbindin-D9k and calbindin-D28k, facilitate diffusion of Ca2+ through the cytosolic compartment of renal and intestinal cells, which transport Ca2+ transcellularly. In the study presented here, parvalbumin, calbindin-D9k, and calbindin-D28k were localized precisely by immunocytochemistry in rat kidney. Antisera recognizing specifically the thick ascending loop of Henle, the connecting tubules and collecting ducts, and the intercalated cells of the collecting ducts were used to identify different cell types. In rat kidney cortex, parvalbumin and calbindin-D9k colocalized in the thick ascending loop of Henle, the distal convoluted tubule, the connecting tubule, and the intercalated cells of the collecting duct. Strikingly, in all responsive cells, parvalbumin and calbindin-D9k were exclusively present in a thin layer along the basolateral membrane. In contrast, calbindin-D28k was only present in the distal convoluted and connecting tubule, where it was evenly distributed through the cytosol. In conclusion, the exclusive localization of parvalbumin and calbindin-D9k at the basolateral membrane of immunopositive renal cells implies their involvement in the regulation of transport processes located in these membranes rather than a role as intracellular Ca2+ buffer and Ca2+ shuttle between the two opposing membranes.     

Tamm-Horsfall protein in patients with kidney damage and diabetes

Tamm-Horsfall protein (THP) is a glycoprotein present abundantly in human urine. It is localized in the thick ascending limb of the loop of Henle (TAL) and the early distal convoluted tubule (DCT). The rate of urinary excretion of THP has been studied in various diabetic groups. It has been postulated that urinary THP may be a useful marker for renal damage. The aim of this study was to compare directly the immunogold localization of THP in diabetic and control kidney tissue specimens with or without kidney damage. Immunogold labeling was performed on archival tissue samples of 34 diabetic and 18 control human kidneys at the light microscope level. Slides were ranked as having a high, moderate or low degree of reaction. The majority of diabetic samples had a slightly lower degree of THP, while patients with known renal dysfunction had lowest THP. Previous studies have found a decreased excretion of urinary THP in diabetics. Our results show that decreased gold labeling is associated with known renal damage and may indicate damage to the thick ascending limb of the loop of Henle and the early distal convoluted tubule, irrespective of presence or absence of diabetes.     

Localization and characterization of renal calcitonin receptors by in vitro autoradiography

Calcitonin receptor binding sites were identified in renal cortex and medulla using the radioligand 125I-salmon calcitonin. Microscopic localization of these receptors revealed binding over medullary and cortical thick ascending limb of the loop of Henle and in distal convoluted tubule. A number of receptor positive cells in the inner medulla were also identified. Characterization of the binding demonstrated a single class of high-affinity binding sites in both the medulla and the cortex with affinity constants of 0.74 +/- 0.09 x 10(9) M-1 and 0.32 +/- 0.05 x 10(9) M-1, respectively, and receptor concentrations of 205 +/- 45 fmol/mg protein and 453 +/- 54 fmol/mg protein, respectively. Competition for 125I-salmon calcitonin binding by a wide range of calcitonin analogs revealed a close correspondence between the reported biological potencies and activities in the current system. The localization of binding sites within the nephron corresponds to the reported localization of calcitonin-stimulated adenylate cyclase activity and suggests that the receptor mediated actions of calcitonin in the kidney utilize cyclic AMP as a second messenger. In addition, the microscopic identification of specific calcitonin receptors helps the delineation of direct actions of this hormone from those which are indirect.