Nephridial Tissue Research Articles

Relationship between renal pathology and glomerular podocyte injury in childhood Henoch-Schönlein purpura nephritis

Objective To explore the relationship between renal pathology and glomerular podocyte injury in childhood Henoch-Schonlein purpura nephritis(HSPN). Methods Renal pathological types of 72 children suffering from HSPN were reviewed during the period of Jan.2008 to Jan.2011 from Nanjing General Hospital of Nanjing Command.In those cases, grade in pathology and immunofluorescence types were acquired.Podocyte injury was classified by electron microscope.The relationship between podocyte injury and grade in pathology was analyzed. Results Pathological findings were classified, grade Ⅱ in 34 cases(47.2%) and grade Ⅲ in 38 cases(52.8%). Glomerular podocyte injury was classified by electron microscope as food processes of podocyte fused extensively in 21 cases; food processes of podocyte fused segmental in 35 cases, food processes of podocyte without fusion in 11 cases.There was without renal glamorous in nephridial tissue by electron microscope in 5 cases.Cases with foot process of podocyte fused extensively were more grade Ⅲ than those cases with foot process fused segmental and without fusion.According to renal immunofluorescence pathology, IgA+ IgM+ IgG type was mostly in cases with foot process of podocyte fused extensively, IgA+ IgG type was mostly in cases with food processes of podocyte fused segmental and IgA type was mostly in cases with food processes of podocyte without fusion. Conclusions In childhood HSPN, renal pathology changed not only expressing on mesangial cell hyperplasia but also podocyte injury.The more seriously on the degree of podocyte injury, more obviously in pathologic change. Key words: Henoch-Schonlein purpura nephritis; Renal pathology; Glomerular podocyte injury; Child

The effects of Kangxianling on renal fibrosis as assessed with a customized gene chip

ObjectiveTo determine the mechanisms by which Kangxianling (KXL) treats renal interstitial fibrosis using a customized gene chip. MethodsTwelve out of 18 specific pathogen-free sprague dawley (SPF SD) rats underwent a unilateral ureteral occlusion. These rats were then randomly assigned into either the model unilateral ureteral obstruction (UUO) or Kangxianling (KXL) group. The other six rats were assigned to the sham-operated group. The UUO and sham-operated groups were given normal saline via intragastric administration, whereas the KXL group was given KXL via intragastric administration. All rats were sacrificed for renal tissue collection (i.e. left nephridial tissue), and the detection of genetic changes with the customized chip. ResultsCompared to the sham-operated group, transforming growth factor-β1 (TGF-β1), Smad2, and Smad3 genes were significantly up-regulated in the UUO group, with >1.5-fold rise (P<0.01). The Smad7 gene was significantly reduced in the UUO versus sham-operated group, with a down-regulation of > 1.5-fold (P<0.01). In the KXL group, TGF-β1 Smad2, and Smad3 genes were significantly reduced compared to the UUO group, with a down-regulation of > 1.5-fold (P<0.01), whereas the Smad7 gene was significantly increased compared to the UUO group, with an up-regulation of > 1.5-fold (P<0.01). ConclusionIt was found that KXL can significantly reduce the gene levels of TGF-β1 Smad2, and Smad3. Immunohistochemistry findings also revealed significantly lower TGF-β1/Smads-mediated gene transcription activity. These findings suggest that KXL may negatively regulate the TGF-β1/Smads signal pathway to inhibit the occurrence of renal fibrosis.

The study of the relationship between the expression of Adrenomedullin and the microvessel density in renal cell cancer

Objective To investigate the relationship between the expression of Adrenomedullin ( ADM) and the microvessel density ( MVD) in renal cell cancer( RCC) . Methods In 33 cases of RCC patients, the ADM expression was detected by immunohistochemistry and flow cytometry ( FCM) , and the MVD was also counted in the tumortissue. Results ADM was expressed in all cases of RCC (33/33,100. 0% ) ,but only 3 cases (3/33,9. 1% ) expressed in the normal nephridial tissue. The ADM expression levels in RCC (1267. 84 ± 127. 52) was higher than that in the normal nephridial tissue (970. 36 ±171. 46) P ＜0. 05. Conclusion The level of MVD was correlated to the expression of ADM. ADM is one of the regulators of the new vessels. Key words: Renal cell cancer; Adrenomedullin; Microvessel density; Immunohistochemistry; Flow cytometry

Study on influence of glucoside Tripterygium total tablets on metabolism in rats by NMR metabonomic technique

To investigate the toxic effects of Glucoside Tripterygium total on rats with nuclear magnetic resonance (NMR)-based metabonomic method. The influence of intragastric administration of Glucoside Tripterygium total suspension at two different doses on endogenetic metabolites in normal rat urine was determined with bio-NMR method then analyzed by pattern recognition technique and partial least-squares discriminant analysis (PLS-DA). Histopathological analysis was carried out. Escalations of concentrations of urinary taurine, TMAO and glucose as well as reductions of concentrations of urinary citrate and 2-oxoglutarate were found by analysis of the 1H-NMR spectra, which was coincident with the result of histopathological analysis. The result of pathological examination indicated that pathologic change was not observed in nephridial tissue, but there were obvious changes in hepatic tissue. The urinary metabomic spectra were closely associated with the hepatic toxicity, which manifested the mitochondrial dysfunctions, the abnormal energy metabolism in TCA cycle as well as the abnormal glucose metabolism.

Correlation between hyaluronic acid, hyaluronic acid synthase and human renal clear cell carcinoma

To study the correlation between hyaluronic acid (HA), hyaluronic acid synthase (HAS) and human renal clear cell carcinoma (RCCC). The expression of three HAS isoforms' gene and HA in 93 RCCC tissues, 27 nephridial tissues by the side of RCCC from two hospitals were measured with Real-Time RT-PCR、Western Blot and immunohistochemical methods and analyzed. All RCCC and adjacent normal tissues expressed three HASs' mRNA & protein; at the mRNA level, both RCCC and adjacent normal tissues, expressed more HAS3 than HAS1 or HAS2, their differences were statistically significant (all P values <0.05); but, at the protein level, all HAS isoforms presented the equivalent expression. Compared with the adjacent non-neoplastic kidney tissues, the expression of all HAS isoforms' mRNA in RCCC tissues were increased evidently and their differences were significant (all P values <0.0001); but at the protein level, only the expression of HAS3 increased evidently (P=0.022). In all adjacent normal tissues, more than 80% renal tubular cells strongly expressed HA, however, only the minority RCCC cases (16/93) presented weakly positive HA staining in few cancer nests (5%-30%), the difference were significant (P<0.0001). In RCCC tissues subgrouped according to clinical stage, pathological grade, lymphatic metastasis or not and distant metastasis or not, the HASs' mRNA & protein differential expression all had no statistical significance (all P values >0.05). Different from other malignancy, HA and HASs (except for HAS3) may not play important roles in the biological progress of human RCCC.

Effects of No.2 Renal Failure Recipe on expressions of cyclooxygenase-2 and -1 mRNAs in rats with chronic renal failure

To observe the effects of No. 2 Renal Failure Recipe (No. 2RFR), a compound traditional Chinese herbal medicine, on expressions of cyclooxygenase-2 (COX-2) and cyclooxygenase-1 (COX-1) mRNAs in rats with chronic renal failure (CRF). A rat model of CRF was successfully established by infarction of approximately two-thirds of the left kidney and removal of the right kidney (ablation/infarction, A/I). Thirty A/I rats were randomly divided into untreated group, celebrex group and No. 2RFR group. Another 10 SD rats were selected as normal control group. After 2-month treatment, the pathology of the nephridial tissue was observed with hematoxylin and eosin straining under a light microscope. Renal function including serum creatinine (SCr) and blood urea nitrogen (BUN) was determined by using an automatic biochemical analyzer. Expressions of COX-2 and COX-1 mRNAs in nephridial tissues were detected by reverse transcription-polymerase chain reaction (RT-PCR). No. 2RFR could significantly decrease the levels of SCr and BUN. Renal function and morphology of CRF rats were ameliorated and the expressions of COX-2 mRNA were decreased significantly in the No. 2RFR group and the celebrex group, but the expressions of COX-1 mRNA had no differences among the four groups. No. 2RFR can improve renal function and reduce glomerular sclerosis and renal fibrosis by inhibiting the over-expression of the COX-2 mRNA.

Influence of ethylbenzene on the levels of mandelic acid and phenylglyoxylic acid in urine, ultrastructure and the expressions of Mitochondrial apoptotic-related proteins in the rat nephridial tissues

To investigate the influence of ethylbenzene on the levels of mandelic acid (MA) and phenylglyoxylic acid (PGA) in urine, the ultrastructure and the expressions of mitochondrial apoptotic-related genes in the rat nephridial tissues. Four groups of 10 males of Sprague-Dawley rats were allocated randomly into four groups: control (C) group, low (L) group, moderate (M) group and high (H) group, and inhaled daily with different doses of ethylbenzene: 0, 433.5 mg/m(3), 4335 mg/m(3), and 6500 mg/m(3) 6 h per day, 5 days per week for 13 weeks. The mandelic acid and phenylglyoxylic acid in the urine was assayed by high performance liquid chromatography. The ultrastructure of nephridial tissue was observed via electron microscope. The protein expression levels of Bax, Bcl-2, cytochrome C, Caspase-9 and Caspase-3 in nephridial tissues were measured by Western blot, respectively. The levels of MA [(0.303 +/- 0.148) mg/L, (0.404 +/- 0.154) mg/L] and PGA [(0.168 +/- 0.104) mg/L, (0.174 +/- 0.092) mg/L] in the urine of M and H groups were significantly higher than that in the control and L group [(0.084 +/- 0.070) mg/L, (0.041 +/- 0.029) mg/L] (P < 0.05, respectively). It has been shown a dose-effect relationship between the contents of MA, PGA and MA + PGA and inhaled ethylbenzene, respectively. The mitochondria of rat nephridial tissue of H group became a compact and vacuolar structure with disorder and loss of cristae. The expression levels of Bax in mitochondria of nephridial tissues of M and H groups were significantly lower than that in the control group (P < 0.05). Caspase-3 expression level in H group was remarkably higher than that in the control group (P < 0.05). Compared with the control group, the expression levels of cytochrome C and Caspase-9 were enhanced, while the expression levels of Bcl-2 were restrained in all ethylbenzene-treated groups (P < 0.05, P < 0.05, respectively). The expression levels of Caspase-3 in M and H groups were significantly higher than that in the control group and L group (P < 0.05). Ethylbenzene can induce apoptosis in the cells of nephridial tissues. The apoptotic mechanism might be involved with up-regulation of Bax, cytochrome C, Caspase-9 and Caspase-3, as well as restraint of Bcl-2. The level of MA and PGA in the rat urine could be a parameter of biological dose in vivo after ethylbenzene inhalation.

Study of rAAV-TK-IRES-ES gene therapy of bladder cancer in vivo

Objective To study the therapeutically effect of rAAV-TIE model in vivo. Meth-ods Cell suspensions of T24 cells were injected into the subcutaneously of right scapular region of nude mice. The nude mice were raised under SPF condition and the xenograft tumor growth was ob-served. Bearing tumor nude mice were randomly divided into 5 groups: rAAV-MCS group, rAAV-Tk group, rAAV-ES group, rAAV-TIE group and control group. Four weeks later of treatment, the nude mice were sacrificed. The xenografts tumors were fixed for HE staining. The liver tissue and ne-phridial tissue were also fixed for HE stain. The blood sample and endostatin concentration was as-sayed by ELISA. Results After 3 weeks of injected with T24 cells on nude mice, 25 showed visible tumor on the injected location. The rate of tumor formation was 93%. After 9 days injected by rAAV-ES, rAAV-TK, rAAV-TIE, the tumor volume were: rAAV-ES group(0.75±0.08)cm3 , rAAV-TK group(0.71±0.11)cm3 , rAAV-TIE group(0.52±0.09)cm3 , rAAV-MCS group(1.27±0.13)cm3 and control group (1.24±0.17)cm3. The microvessel density in the different groups were as follow-ings: rAAV-ES group(18.72±2.53)/HP, rAAV-TK group(21.74±4.62)/HP, rAAV-TIE group (12.73±1.78)/HP, rAAV-MCS group(52.38±6.46)/HP and control group(49.94±7.17)/HP. The endostatin concentration in the diffcrent groups were as followings: rAAV ES group(38.52 6.53)μg/L and rAAV-TIE group(40.33±7.48)μg/L. HE staining confirmed the tumor. The liver tissue and kidncy tissue of each group had no obviously cell degeneration or necrosis. Conclusion The rAAV-TIE could inhibit tumor induced angiogenesis and suppress both the initiation and the subse-quent growth of human bladder cancer in nude mice model. Key words: Bladder neoplasms; Adeno-associatcd virus; Suicide gcne; Endostatin

Effect of heme oxygenase-1 on the kidney during septic shock in rats

The aim of this study was to clarify the effect of heme oxygenase (HO)-1 on the kidney during septic shock in rats. Eighty healthy and clean Sprague-Dawley rats were randomly divided into 4 groups: control group (group C) , septic shock group (group SS) , lipopolysaccharide plus ZnPP-IX group (group LZ) , and ZnPP-IX (group Z) . The plasma levels of COHb, Cr, and BUN; the urine levels of gamma-GTP, alpha1-MG, RBP, and NAG; and MDA content, SOD activity; HO-1mRNA; HO-2mRNA; HO-1 protein; and HO-2 protein from kidney were measured. The plasma levels of Cr and BUN; the urine levels of gamma-GTP, alpha1-MG, RBP, and NAG; and MDA content from kidney in group SS were obviously higher than in group C and Z (both P < 0.05), respectively, but lower than in group LZ (both P < 0.05), respectively. In contrast, the plasma levels of COHb and SOD activity from kidney in group SS were obviously lower than those in group C and Z (both P < 0.05), respectively, but higher than in group LZ (both P < 0.05), respectively. The HO-1mRNA and HO-1 protein of nephridial tissue in group LZ were obviously higher than in group C and Z, respectively (both P < 0.05), but lower than in group SS, respectively (both P < 0.05). There were no significant differences of HO-2mRNA and HO-2 protein among these 4 groups (both P > 0.05). It is concluded that upregulation of the HO-1 protein might contribute to the protection of kidney during septic shock in rats.

Effects of puerarin on receptor for advanced glycation end products in nephridial tissue of streptozotocin-induced diabetic rats

To explore the effects of puerarin on mRNA expression of advanced glycation end products (AGE) specIfic cellular receptor (RAGE) in renal cortex of diabetic rats induced by streptozotocin (STZ). We induced Diabetic rats by an intraperitoneal injection of STZ in Sprague-Dawley (SD) rats. 30 male SD rats were randomly divided into 3 groups, diabetes adding puerarin group (DP group, n = 11, intraperitoneal injection of puerarin 100 mg/kg d), Diabetes group (D group, n = 11) and normal control group (C group, n = 8). The body weight (BW) and blood glucose (BG) were measured every 2 weeks. eight weeks later, all rats were sacrificed and the expression of RAGE mRNA was detected in renal cortex by reverse transcription-polymerase chain reaction (RT-PCR), respectively, and renal AGEs content was determined by fluorescence microscopy. Compared with those of control group, the BW and BG were lower in DP group and D group at 8th week (P < 0.01). RAGE/beta-actin ratio were 0.263 +/- 0.023, 0.435 +/- 0.010, 0.141 +/- 0.045, respectively, in DP group, D group and C group, and there was significant difference between every two groups (P < 0.01). The renal AGEs fluorescence intensity of DP group was weaker than D group, stronger than C group. Puerarin can protect the renal tissue from the impairment of hyperglycemia and AGE by decreasing AGEs contents and inhibiting of the expression of RAGE mRNA in the kidney.

Effect of Panaxnotoginseng on the Platelet-derived Growth Factor-BB and Its mRNA's Expression on Adriamycin-induced-nephropathy Rats

Abstract Objective To investigate the change of platelet-derived growth factor-BB (PDGF) in adriamycin nephropathy rats, and to study the effect of Panaxnotoginseng on adrimysin-induced nephropathy rats and its possible mechanism Methods Rats with unilateral nephrectomy or intraperitoneal injection of adriamycin-induced nephropathy were used. 45 Wistar rats were randomly divided into 5 groups: normal control group, adriamycin nephropathy group, sham operation group, Lotensin treated group, and Panaxnotoginseng treated group. The Renal tissues were examined by microscopy at the 10th week after operation. Immunohistochemistry and in-situ hybridization were used to measure the expression of PDGF protein and PDGFmRNA in the nephridial tissue Results The expression of PDGF protein and PDGFmRNA in the normal and sham operation group is low in the intracytoplasm of mesangial cells, renaltubular epithelial cells, and renal tubular epithelial cells. Compared with sham operation group, the expression of PDGF protein and PDGFmRNA as yellow particulate in the mode group is markedly increased, and the fibrosis of nephridial tissue is obvious. Panaxnotoginseng can significantly decrease the expression of PDGF protein and PDGFmRNA (p Conclusions Panaxnotoginseng may suppress the development of fibrosis, at least in part via down-regulating the expression of protein and mRNA of PDGF.

Preparation of chitosan-polyaspartic acid-5-fluorouracil nanoparticles and its anti-carcinoma effect on tumor growth in nude mice

To prepare chitosan-polyaspartic acid-5-fluorouracil (CTS-Pasp-5Fu) nanoparticles and investigate its anti-carcinoma effect and toxicity. CTS-Pasp-5Fu nanoparticles were synthesized by ionic gelatification. Male BABL/c nude mice were injected with SGC-7901 gastric carcinoma cell line mass to establish a human gastric carcinoma model. They were randomly allocated into 4 groups: CTS-Pasp-5Fu (containing 5-Fu 1.25 mg/kg), 5-Fu (1.25 mg/kg), CTS-Pasp and normal saline groups. Tumor weight was measured and assay of colony forming unit-granulocyte and macrophage (CFU-GM) was performed. The structural change of cells and tissues was observed and the Bax and Bcl-2 genes were detected. Compared with normal saline, the inhibition rates of tumor growth for the CTS-Pasp, 5-Fu and CTS-Pasp-5Fu groups were 5.58%, 58.69% and 70.82%, respectively. The tumor inhibition rates for the CTS-Pasp, 5-Fu and CTS-Pasp-5Fu groups were 5.09%, 65.3% and 72.79%, respectively. There was a significant decrease in the number of CFU-GM formation and increase of total bilirubin, and alanine aminotransferase in the 5-Fu group, but no change in those of the other three groups. There was no change in white blood cell count and creatinine among the four groups. Pathological section of liver and nephridial tissues showed that the damage in the 5-Fu group was more severe than that in the CTS-Pasp-5Fu group. 5-Fu and CTS-Pasp-5Fu groups could both down-regulate the Bcl-2 expression and up-regulate the Bax expression to different extent, and the accommodate effect of CTS-Pasp-5Fu was more obvious than 5-Fu. The tumor inhibition rate of CTS-Pasp-5Fu nanoparticles is much higher than that of 5-Fu alone.

Accumulation of Calcium in the Antennal Gland during the Molting Cycle of the Freshwater Crayfish Procambarus clarkii

Calcium (Ca+2) was histochemically localized in antennal glands during the molting cycle of the crayfish Procambarus clarkii. In tissues involved in electrolyte reabsorption, alizarin red S formed precipitates with Ca+2 (4-30 iim in diameter). In the coelomosac, the number of Ca+2-alizarin precipitates did not vary significantly during the molting cycle. In labyrinth and nephridial canal tissues, Ca+2 precipitates were significantly more abundant during postmolt than during intermolt, with a maximum at 4 days postmolt; by 14 days postmolt, Ca+2 precipitation had decreased in the labyrinth and nephridial canal, although t he values remained significantly greater than intermolt levels. Distal nephridial canal cells treated with potassium oxalate contained mitochondria with electron-dense precipitates (30-85 nm in diameter). These deposits reached a maximum at 4 days postmolt, but remained significantly above intermolt levels by 14 days postmolt. These findings suggest that Ca+2 accumulates in reabsorptive tissues during postmolt and that the mitochondria of distal nephridial canal cells may also serve to maintain intracellular and extracellular Ca+2 homeostasis. Additional key words: nephridial canal, labyrinth, alizarin red S, K+-oxalate, histochemistry The molting cycle of a freshwater crayfish can be used as a model system to study cellular mechanisms of calcium transport associated with cuticular calcification and decalcification. At intervals, the old cuticle is decalcified, and a new cuticle is formed underneath. Once the old cuticle is shed at ecdysis, the new cuticle undergoes calcification. During the molting cycle, these animals experience physiological challenges related to ionic regulation that are more pronounced in freshwater than in saltwater, specifically, low calcium

Cardiac output and tissue blood flow in the abalone, Haliotis cracherodii (Mollusca, Gastropoda)

Little is known about the characteristics of hemolymph (blood) flow in animals with open circulatory systems. We measured cardiac output and blood flow to specific tissues in the black abalone, Haliotis cracherodii, a gastropod mollusk. The use of thermodilution allowed us to make repeated measurements of cardiac output and cardiac stroke volume over relatively short time intervals (5-10 heart beats) in resting, unrestrained abalone while disturbing the animals minimally. Anatomical studies of the abalone circulation showed that the arterial system terminated in small diameter (approaching 10-20 micron in some cases) lacunar tissue spaces. Because of this, we used radioactive microspheres (which must be trapped in the tissue vasculature) to measure blood flow rates to selected tissues. The major findings of our study were that 1) cardiac output in the black abalone ranged from about 100 to 150 ml X kg-1 X min-1, and was highly correlated with body size; 2) weight-specific cardiac stroke volume was about 5 ml X kg-1, considerably larger than that of a mammal; 3) tissue blood flow rates ranged from 10 ml X 100 g-1 X min-1 (foot muscle) to 80 ml X 100 g-1 X min-1 (nephridial tissue), similar to typical tissue blood flow rates in mammals. Our data suggest that the blood in the abalone is directed to the tissues not in proportion to percent total body weight the tissues represent (as might be expected in an open vascular system), but apparently in proportion to tissue metabolic rate.

Morphology and fine structure of two marine tubificids (Oligochaeta), closely related to the gutlessPhallodrilus spp.

The morphology and ultrastructure of the two interstitial marine tubificidsPhallodrilus prostatus andAktedrilus monospermathecus (Oligochaeta), closely related to the gutlessPhallodrilus spp., is described. Since pertinent histological descriptions of ‘regular’ marine tubificids were found to be non-existent, this study provides a structural basis allowing comparisons both with limnetic species and the recently described marine anenteric forms. Compared to the few freshwater tubificids investigated histologically, major differences are the substitution of the dorsal blood vessel by a large sinus, the close contact between nephridial tissue and the subintestinal vessel, a characteristic ‘wavy’ arrangement of cuticular collagen fibres, and the formation of branched epicuticular projections. The structural divergences from the gutless relatives refer mainly to the body wall, the vascular system and its endothelial structure. The various peculiarities outlined here indicate that generalizations of the situation as found in the few histological studies are as yet problematical.

Memoirs: On the Structure of the Nephridia and ‘funnels’ of the Indian Leech Hirudinaria with remarks on these organs in Hirudo

1. The nephridial system of Hirudinaria consists of a series of seventeen pairs of nephridia metamerically arranged in somites VI to XXII. The first sis pairs occur in the pretesticular segments (VI-XI), while the remaining eleven pairs lie in the testicular segments (XII-XXII). 2. A typical nephridium consists of the following parts: (i) the initial lobe, (ii) the apical lobe, (iii) the inner lobe, (iv) the main lobe, (v) the vesicle-duct and the vesicle. All the nephridia in the testicular region possess ‘funnels’ (ciliated organs) which are enclosed within the ampullae of the perinephrostoniial sinus. There is no continuity or connexion of these ‘funnels’ with the nephridia in the adult leech. 3. The inner end of the initial lobe is directed towards the testis-sac and either ends freely within the connective tissue without coming in contact with the testis-sac, o r- is embedded in fibrous tissue in external contact with the wall of the sac, or becomes incorporated within the outer wall of the perinephrostomial sinus. 4. The initial lobe (testis-lobe) forms a very long coiled string of cells round the apical lobe and part of the inner lobe. The inner lobe (the ‘recurrent lobe’ of Bourne) forms a distinct strip of nephridial tissue enclosed between the two limbs of the main lobe besides a small piece which runs alongside the apical lobe. The inner lobe canals serve to connect the intra-cellular canals of all the lobes with one another. 5. The cells of the different lobes of the nephridium are tunnelled through by intra-cellular canals and canaliculi which form a continuous branching network throughout the body of the nephridium. Besides, there is an intra-cellular central canal which makes 1 3/4 ‘rounds’ through the various lobes of the nephridium and opens into the vesicle. The intra-cellular canals and canaliculi open directly or indirectly into the central canal. 6. The vesicle has no muscular layer, and its wall is not contractile. The evacuation of the contents of the vesicle is brought about by the contraction of ventro-lateral mameles of the bodywall that extend across all the resides. The vesicle and the terminal excretory duet do not develop from the rudiments of the true nepfaritliam, but are formed from an ingrowth of the epidermis. 7. A fully developed adult ‘funnel’ (ciliated organ) is a compound structure consisting of (1) a central reservoir and (2) a large number of small independent funnels set on the reservoir and opening into it. The funnels are profusely dilated. Each funnel is composed of five to six cells, and has the appearance of an ear-lobe with a broad distal and a narrow proximal end. 8. The reservoir is the seat of manufacture of corpuscles which are thrown out of the reservoir through the funnels into the surrounding sinus by the active movements of the cilia of the numerous funnels. 9. The ‘funnel’ is not a degenerate structure. It has, infact, multiplied into numerous small ciliated funnels, which, are much more effective in their ciliary action than a single funnel, even of a large size, could be. Cilia of the funnels show very vigorous movements which keep the fluid in the sinus in constant active circulation. The ciliated organ, instead of serving a renal excretory function, has here become subservient to the sinus-system. 10. The botryoidal vessels are in direct communication with the perinephrostomial sinus. Possibly the corpuscles take up pigment and become the chloragogen cells in the botryoidal vessels. 11. In the embryonic condition the ‘funnel’ is a solid mass of cells which is distinctly continuous with the nephridinm by means of a delicate strand of cells. This connexion of the ‘funnel’ with the nephridium snaps later, and the two become discontinuous and discrete structures. In the embryonic solid ciliated organ the funnel-forming cells can be clearly distinguished from the cells of the reservoir. The ‘funnel’ becomes enclosed at an early stage in the perinephrostomial sinus, which is a part of the reduced coelom. 12. The nephridial system of Hirudo is essentially similar to that of Hirudinaria. In Hirudo the initial lobe does not coil round the apical lobe, but forms one mass round the ampullae of the perinephrostomial sinus and another between the apical and main lobes. The inner end of the nephridium is closed as in Hiradinaria. The ‘funnels’ have the same structure and perform the same function as in Hirudinaria.