Regulation of renal urea transporters

Urea is important for the conservation of body water due to its role in the production of concentrated urine in the renal inner medulla. Physiologic data demonstrate that urea is transported by facilitated and by active urea transporter proteins. The facilitated urea transporter (UT-A) in the terminal inner medullary collecting duct (IMCD) permits very high rates of transepithelial urea transport and results in the delivery of large amounts of urea into the deepest portions of the inner medulla where it is needed to maintain a high interstitial osmolality for concentrating the urine maximally. Four isoforms of the UT-A urea transporter family have been cloned to date. The facilitated urea transporter (UT-B) in erythrocytes permits these cells to lose urea rapidly as they traverse the ascending vasa recta, thereby preventing loss of urea from the medulla and decreasing urine-concentrating ability by decreasing the efficiency of countercurrent exchange, as occurs in Jk null individuals (who lack Kidd antigen). In addition to these facilitated urea transporters, three sodium-dependent, secondary active urea transport mechanisms have been characterized functionally in IMCD subsegments: (1) active urea reabsorption in the apical membrane of initial IMCD from low-protein fed or hypercalcemic rats; (2) active urea reabsorption in the basolateral membrane of initial IMCD from furosemide-treated rats; and (3) active urea secretion in the apical membrane of terminal IMCD from untreated rats. This review focuses on the physiologic, biophysical, and molecular evidence for facilitated and active urea transporters, and integrative studies of their acute and long-term regulation in rats with reduced urine-concentrating ability.     

Dysregulation of renal sodium transporters in gentamicin-treated rats

We aimed to investigate the molecular mechanisms underlying the renal wasting of Na(+), K(+), Ca(2+), and Mg(2+) in gentamicin (GM)-treated rats. Male Wistar rats were injected with GM (40 or 80 mg/kg/day for 7 days, respectively; GM-40 or GM-80). The expression of NHE3, Na-K-ATPase, NKCC2, ROMK, NCC, alpha-, beta- and gamma-ENaC, and CaSR was examined in the kidney by immunoblotting and immunohistochemistry. Urinary fractional excretion of Na(+), K(+), Ca(2+), and Mg(2+) was increased and urinary concentration was decreased in both GM-40 and GM-80 rats. In cortex and outer stripe of outer medulla (cortex) in GM-80 rats, the expression of NHE3, Na-K-ATPase, and NKCC2 was decreased; NCC expression was unchanged; and CaSR was upregulated compared to controls. In the inner stripe of outer medulla (ISOM) in GM-80 rats, NKCC2 and Na-K-ATPase expression was decreased, whereas CaSR was upregulated, and NHE3 and ROMK expression remained unchanged. In GM-40 rats, NKCC2 expression was decreased in the cortex and ISOM, whereas NHE3, Na-K-ATPase, CaSR, ROMK, and NCC abundance was unchanged in both cortex and ISOM. Immunoperoxidase labeling confirmed decreased expression of NKCC2 in the thick ascending limb (TAL) in both GM-80- and GM-40-treated rats. Immunoblotting and immunohistochemical analysis revealed increased expression of alpha-, beta-, and gamma-ENaC in cortex in GM-80 rats, but not in GM-40 rats. These findings suggest that the decrease in NKCC2 in TAL seen in response to low-dose (40 mg/kg/day) gentamicin treatment may play an essential role for the increased urinary excretion of Mg(2+) and Ca(2+), and play a significant role for the development of the urinary concentrating defect, and increased urinary excretion of Na(+) and K(+). At high-dose gentamicin, both proximal and TAL sodium transporter downregulation is likely to contribute to this.     

Massive reduction of urea transporters in remnant kidney and brain of uremic rats

BACKGROUND: The facilitated urea transporters (UT), UT-A1, UT-A2, and UT-B1, are involved in intrarenal recycling of urea, an essential feature of the urinary concentrating mechanism, which is impaired in chronic renal failure (CRF). In this study, the expression of these UTs was examined in experimentally induced CRF. METHODS: The abundance of mRNA was measured by Northern analysis and that of corresponding proteins by Western blotting in rats one and five weeks after 5/6 nephrectomy (Nx). RESULTS: At five weeks, urine output was enhanced threefold with a concomitant decrease in urine osmolality. The marked rise in plasma urea concentration and fall in urinary urea concentration resulted in a 30-fold decrease in the urine/plasma (U/P) urea concentration ratio, while the U/P osmoles ratio fell only fourfold. A dramatic decrease in mRNA abundance for the three UTs was observed, bringing their level at five weeks to 1/10th or less of control values. Immunoblotting showed complete disappearance of the 97 and 117 kD bands of UT-A1, and considerable reduction of UT-A2 and UT-B1 in the renal medulla. Similar, but less intense, changes were observed at one-week post-Nx. In addition to the kidney, UT-B1 is also normally expressed in brain and testis. In the brain, its mRNA expression remained normal one-week post-Nx, but decreased to about 30% of normal at five-weeks post-Nx, whereas no change was seen in testis. CONCLUSIONS: (1) The decline in urinary concentrating ability seen in CRF is largely due to a major reduction of UTs involved in the process of urea concentration in the urine, while factors enabling the concentration of other solutes are less intensely affected. (2) The marked reduction of brain UT expression in CRF may be responsible for brain edema of dialysis disequilibrium syndrome observed in some patients after fast dialysis.     

Osmoregulation of aldose reductase and sorbitol dehydrogenase in cultivated interstitial cells of rat renal inner medulla.

BACKGROUND: Little is known about sorbitol metabolism in renal papillary interstitial cells. For characterization we studied regulation of sorbitol synthesis by aldose reductase (AR) and degradation by sorbitol dehydrogenase (SDH) in papillary interstitial cells. METHODS: Interstitial cells were isolated from rat renal inner medulla to a pure cell fraction. mRNA was isolated from cultivated cells and sorbitol, AR and SDH activity were determined enzymatically in homogenates. RESULTS: Sorbitol concentration in these cells at 300 mosmol/l was 4.4+/-0.3 vs 78+/-3.6 micro mol/g protein at 600 mosmol/l. At steady-state conditions at 300 mosmol/l, AR activity was nearly the same as SDH activity (15.1+/-1.6 vs 16.6+/-2.0 U/g protein). At 600 mosmol/l, AR activity increased to 82.5+/-11.4 U/g protein and SDH activity to 31.5+/-6.0 U/g protein. Studying the time course of enzyme activity after changing osmolarity from 300 to 600 mosmol/l, we found half maximal stimulation after 2-3 (AR) or 3 (SDH) days. The amount of AR-mRNA preceded the rise of enzyme activity, whereas SDH-mRNA was not significantly influenced. Lowering osmolarity from 600 to 300 mosmol/l, enzyme activity decreased to less than half within 2 (AR) or 1 (SDH) day(s). CONCLUSIONS: The results suggest that sorbitol metabolism contributes to handling of osmotic stress in rat renal papillary interstitial cells.     

Effect of NaCl and urea concentration comparable to renal medulla on superoxide production by human polymorphonuclear leukocytes

Summary The influence of hyperosmolarity on superoxide production by polymorphonuclear leukocytes (PMNL) was examined using NaCl and urea as osmotic substances. Superoxide production was inhibited in a hyperosmotic environment produced by high concentrations of these substances with the following IC₅₀:440±75 (SD) mOsm/kg for NaCl and 660±100 for urea. In the case of NaCl, this inhibition was time-dependent and abolished at 4°C. Since PMNL pump out Na⁺ ion for maintenance of cellular volume in an energy dependent fashion, it was suggested that the inhibition of superoxide production was due to the exhaustion of energy stores. On the other hand, urea inhibition was almost immediate and remained even when preincubation was performed at 4°C. Because the transport of urea through the cell membrane is known to be energy independent, these findings suggested that urea was either an inhibitor of the NADPH oxidase or a scavenger of superoxide anion.     

Regulation of atrial natriuretic peptide-stimulated cGMP production in the inner medulla.

Studies were performed to examine the regulation of atrial natriuretic peptide- (ANP) stimulated guanylate cyclase in the the inner medulla. Primary cultures of rat inner medullary collecting tubular cells exposed to 10(-7) M ANP increased cGMP formation to 31.2 +/- 1.8 compared to the basal production of 2.1 +/- 0.6 fm/micrograms protein. This response did not appear to be transduced via a Gi protein, as preincubation with pertussis toxin did not alter the response to 10(-7) M ANP, and saponized cells exposed to 10 microM GTP gamma S did not enhance the response to ANP (77.3 +/- 5.9 vs. 86.7 +/- 6.3 g/micrograms). Likewise, changes in extracellular Ca2+ from 0.5 to 3.0 mM, decrements in intracellular Ca2+ with EGTA or increments in intracellular Ca2+ with ionomycin (5 microM) did not significantly alter the response to ANP. Neither activation of protein kinase A with forskolin (36.5 +/- 5.1) nor of protein kinase C with s,n-1,2-dioctanoylglycerol (33.2 +/- 2.5) altered the response to 10(-7) M ANP (32.2 +/- 3.3, NS). As the inner medullary environment was hypertonic, the effect of altering tonicity was studied. Cells grown for 48 hours in hypertonic media (600 mOsm/kg H2O) displayed enhanced response to 10(-8) and 10(-7) M ANP when osmolality was raised by either Na+ alone or in combination with urea, but not by urea alone. Our studies demonstrate that ANP-stimulated guanylate cyclase is insensitive to alterations in either intra- or extracellular Ca2+, is not subject to inhibition by protein kinase, and does not involve a pertussis-sensitive G protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Angiotensin II: breathtaking in the renal medulla

Angiotensin II (Ang II) is a major contributor to the progression of renal fibrosis. Wang and colleagues provide evidence that signaling through the prolyl-4-hydroxylase domain (PHD)-hypoxia-inducible factor-1 (HIF-1) pathway mediates profibrotic effects of Ang II in rat renal medullary interstitial cells under normoxic conditions, thus placing the HIF oxygen-sensing pathway into the center of an Ang II-induced profibrotic signaling cascade.     

Effect of long-term type 1 diabetes on renal sodium and water transporters in rats

The effects of long-term diabetes in the presence of established nephropathy on tubular function remains poorly understood. We evaluated the levels of the main sodium and water transport proteins expressed in the kidney after long-term (8 weeks) of streptozotocin (STZ)-induced type 1 diabetes mellitus (DM) in untreated (D) and insulin (4 U/s.c./day)-treated (D+I) rats. D animals presented upregulation ( approximately 4.5-fold) of Na/glucose cotransporter (SGLT1), whereas the alpha-subunit of the epithelial sodium channel (alpha-ENaC) and aquaporin 1 (AQP1) were downregulated ( approximately 20 and 30% respectively) with no change in the Na/H exchanger (NHE3), Na/Cl cotransporter (TSC) and AQP2. Insulin replacement partially prevented these alterations and caused increases in the expression of alpha-ENaC and AQP2. These effects suggest an action of insulin in the tubular transport properties. The upregulation of SGLT1 may constitute a mechanism to prevent greater glucose losses in the urine but it may result in glucotoxicity to the proximal epithelial cells contributing to the diabetic nephropathy. The decrease of alpha-ENaC in D animals may compensate for the increased sodium reabsorption via SGLT1 resulting in discrete natriuresis. DM-induced polyuria was not due to changes in AQP2 expression.     

Red cell trapping and postischemic renal blood flow. Differences between the cortex, outer and inner medulla.

The distribution of blood flow in the rat kidney after 60 minutes of renal ischemia was studied by single-fiber laser-Doppler flowmetry. Blood flow in superficial cortex and inner medulla was measured with a probe directed towards the kidney surface and exposed papilla, respectively. Outer medullary blood flow was measured with a probe introduced through the renal core. After ischemia the blood flow decreased to 60% of the preischemic value (P less than 0.01) in superficial cortex and to 16% (P less than 0.01) in outer medulla, while inner medullary blood flow increased paradoxically to 125% (P less than 0.01). There was extensive trapping of red blood cells (RBC) in the outer medulla, but not in the inner medulla or cortex. The fractional RBC volume as measured by radiolabeled RBCs was 21% in the inner stripe of the outer medulla, but 2% in this area in a normal kidney. To investigate the influence of RBC trapping on intrarenal distribution of blood flow after ischemia, the hematocrit was reduced from 46% to 31% by isovolemic hemodilution. When performed before ischemia, this maneuver almost completely abolished RBC trapping. In this group blood flow in both outer and inner medulla was almost unchanged after ischemia, while superficial cortical blood flow decreased to 66% (P less than 0.01) of the pre-ischemic value. It is concluded that RBC trapping in the outer medulla causes a large decrease in blood flow in this area and, at the same time, shunting of blood to the inner medulla. In the absence of RBC trapping, blood flow of both outer and inner medulla is well preserved after ischemia.     

高盐饮食对自发性高血压大鼠肾髓质环氧化酶2表达的影响

目的观察自发性高血压大鼠（SHR）肾髓质环氧化酶2（COX2）的表达以及不同盐负荷状态下COX2的变化。方法6周龄SHR大鼠及其对照Wistar-Kyoto（WKY）大鼠各12只，随机分为低盐组和高盐组（WKY-LS组，WKY-HS组，SHR-LS组，SHR-HS组），分别给予高盐（含8％NaCl）或低盐（含O．04％NaCl）饮食3d。观察基础状态及不同盐负荷前后大鼠尾动脉血压、24h尿量（UV）、尿钠（UNa）以及COX2代谢物6k．PGFlα排泄情况。应用免疫组化和Western印迹的方法检测肾髓质COX2蛋白表达。结果给予不同盐负荷3d后，SHR-Hs组血压显著升高[（12．59±5．13）比（11．94±3．76）mmHg，P〈0．05]。基础状态及不同盐负荷状态下，SHR大鼠24hUV、UNa排泄与WKY大鼠相比均显著下降（P均〈O．05）。与各自基础状态相比，低盐饮食后两种大鼠的UV和UNa排泄显著降低；高盐饮食后则显著增加（P均〈O．05）。SHR和WKY大鼠尿COX2代谢物6k-PGFlα的排泄对盐负荷的反应也类似，高盐组均较低盐组显著增加（P〈0．05），但同样盐负荷状态下两组之间差异无统计学意义。基础状态下及低盐饮食SHR与WKY大鼠肾髓质COX2表达差异无统计学意义；给予高盐饮食后WKY大鼠肾髓质COX2表达增加2．06倍，SHR大鼠增加1．87倍，但两种大鼠之间差异无统计学意义。结论SHR大鼠肾脏水盐排泄功能受损，高盐饮食后血压进一步升高，水钠潴留，同时伴有肾髓质COX2表达升高，但不同盐负荷对SHR大鼠肾髓质COX2表达的影响与WKY大鼠相同。提示肾髓质COX2参与了水盐代谢的调节，且在SHR大鼠中表达正常，但并非该动物模型高血压形成及水钠潴留的关键因素。     

A biologic role of HIF-1 in the renal medulla

BACKGROUND: Activation of hypoxia-inducible factor-1 (HIF-1) is the primary defensive mechanism against hypoxia. HIF-1 activation generally occurs in pathologic disruption of tissue oxygenation. However, a biologic role of HIF-1 in the medulla of the kidney, which is considered perpetually hypoxic under physiologic conditions due to its unique circulation, remains to be elucidated. METHODS: The expression of HIF-1alpha was detected by immunohistochemical analysis. Functional studies of HIF in medulla were carried out by gene transfer of various plasmids by retrograde injection via ureter. RESULTS: Our immunohistochemical analysis detected HIF-1alpha in the inner stripe and the inner medulla of normal rats. Water deprivation increased the number of HIF-1alpha-positive cells, which may be mediated by an increase in medullar workload and a decrease in local blood flow. To perform functional studies, we performed gene transfer. Efficient expression of the transgene was confirmed using an enhanced green fluorescent protein (E-GFP) expressing vector. Our histologic and immunoblotting analysis detected the transgene product at the inner medulla and the inner stripe 48 hours after injection. Administration of negative-dominant HIF induced severe damage in the medulla of normal rats. In contrast, gene transfer of constitutively active HIF (HIF/VP16) induced expression of various HIF-regulated genes and protected the medulla against ischemic insults. CONCLUSION: Our studies demonstrated a crucial role of HIF in the renal medulla under normal and hypoxic circumstances.     

The renal medulla in acute renal allograft rejection: comparison with renal cortex

A retrospective cohort study was undertaken to evaluate thediagnostic value of the renal medulla in acute renal allograftrejection (ARAR). One hundred and ninety-five biopsies from98 patients were randomly selected out of 565 transplant biopsies.Biopsies were graded blindly from Grade 0 (no rejection) toGrade 3 (severe rejection) using standard criteria; ARAR wasconfirmed by a fall in all cases of mean serum creatinine concentrationfrom 0.331 ± 0.182 to 0.184 ± 0.079 mmol/l, withanti-rejection therapy. In the 43 biopsies which contained bothcortex and medulla, the ARAR grades and the intensities of mononuclearcell, plasma cell, polymorphonuclear cell and eosinophil infiltrates,and of interstitial oedema and haemorrhage, were similar incortex and medulla (Spearman's Rank Correlation r=0.55–0.81,P < 0.001 ). The sensitivity, specificity and overall accuracyof medullary changes in predicting ARAR changes in the cortexwere 77%, 100% and 38%, respectively. Acute vascular rejectionchanges could not be compared between renal cortex and renalmedulla because of the anatomical differences between cortexand medulla. Further evaluation of ARAR in the all 195 biopsies,of which 188 had cortical tissue and 50 had medullary tissue,showed no significant differences in histological features (P> 0.05), except for more cortical biopsies with plasma cells(29%) than medullary biopsies with plasma cells (10%; P <0.02). It is concluded that: (1) ARAR histological changes aresimilar in cortex and medulla; (2) the predictive value of ARARmedullary changes for cortical rejection changes has low sensitivity(77%) and high specificity (100%). It is suggested that a predominantlynormal medullary renal biopsy in suspected rejection shouldbe repeated to obtain cortical tissue.     

Organic solute profiles and transport in the rat renal medulla

Renal inner medullary cells accumulate high concentrations of organic solutes. Sorbitol and glycerophosphorylcholine accumulation (concentration) increase progressively from the outer medulla to the papillary tip. Inositol accumulation is the reverse; its concentration decreases from the outer medulla to the papillary tip. Diabetes mellitus (1 week) increases sorbitol at all levels of the inner medulla, decreases glycerophosphorylcholine and has little effect on inositol. Thin slices of the inner medulla incubated in vitro concentrate inositol and choline from the medium. Although the inner medullary cells are permeable to sorbitol, membrane transport appears to play no role in its accumulation.